Inhibitory effect of different probiotic formulations on Salmonella Heidelberg, avian pathogenic Escherichia coli and Campylobacter jejuni

A wide variety of microorganism species have been used as probiotics to improve the intestinal microbial balance, control and prevent the colonization of pathogenic bacteria and promote growth in animal production. Based on the various beneficial factors of probiotic agents, this study aimed to evaluate the effectiveness of three different formulations of probiotics strains through the analysis of antagonistic capacity against common pathogens from chickens gastrointestinal tract. Three formulations composed by Bacillus, Lactobacillus, Saccharomyces, Enterococcus, Bifidobacterium and Pediococcus were tested in vitro by the method of probiotic culture spot in plates seeded with inocula of Escherichia coli, Salmonella Heidelberg and Campylobacter jejuni. The inhibition halos were measured and classified according to the degree of pathogenic bacteria inhibition. The formulation containing an association among Bacillus, Lactobacillus and Saccharomyces presented better results when compared to the other formulations.

Campylobacter fetus in abomasal fluid from spontaneously aborted bovine and ovine fetuses

Pregnancy losses are a major concern in livestock industry due to their economic impact on producers. Campylobacter fetus subspecies fetus (Cff) and C. fetus subspecies venerealis (Cfv) are directly related to reproductive failures in ruminants. Cff colonizes the gastrointestinal tract of a wide range of hosts leading to abortion, while Cfv is restricted to genital tract being generally associated to infertility in bovine. Considering the great economic losses related to campylobacteriosis in cattle and ovine herds, this study aims to investigate the occurrence of C. fetus, considering Cff and Cfv subspecies, in bovine and ovine spontaneously aborted fetuses in state of Rio Grande do Sul, Brazil. In this study, samples of abomasal fluid collected from 30 spontaneously aborted bovine (n = 18) and ovine (n = 12) fetuses were investigated for the detection of Campylobacter fetus throughout conventional PCR. Positive fetuses for C. fetus presence were further analyzed by molecular assays for Cff and Cfv detection, in order to determine subspecies identification. When available, samples of the main organs of the thoracic and abdominal cavities, as well as the brain, skeletal muscle, eyelid, skin, and placenta were collected for further histopathological analyses and bacterial culture, aiming to assess the presence of infection lesions and pathogens in those sites, respectively. Additionally, RT-qPCR assays were also performed for the detection of ruminant pestivirus, in order to detect bovine viral diarrhea cases. Throughout the present methodology, C. fetus was detected in the abomasal fluid samples of 2 bovine fetuses, being both identified as Cfv subspecies by PCR. Histopathological analyses demonstrated that macroscopic and microscopic changes found in the Cfv-positive animals were not either specific or directly related to Campylobacter infections. Moreover, no significant bacterial growth was observed in microbiological culture from the collected tissues, and both fetuses were negative for ruminant pestivirus. Differently, there was no detection of C. fetus in any of the analyzed ovine fetuses. Considering that abortion diagnosis rates reported in cattle and sheep industry are highly variable among the published studies, and that abortion diagnoses are commonly inconclusive due to difficulties in sampling methodology and inadequate identification of the pathogen involved, it is important to investigate the etiological causes of abortion the herds for better understanding the causes of pregnancy issues and monitoring their occurrence. In addition, the absence of pathognomonic lesions in the tissues investigated in the histopathological analyses observed in this study strongly suggests that well-known etiological agents commonly associated to abortion, such as Leptospira spp., Toxoplasma spp., Chlamydia spp. and Neospora caninum, are unlikely to be the cause of infection of the analyzed fetuses. Taking this into account, the presence of C. fetus in the abomasal fluid samples from two bovine fetuses demonstrated in the present study suggests the possible association of Cfv not only with infertility, but also with cases of bovine abortion, highlighting the importance of investigating unusual causal agents of abortions in sheep and cattle. Overall, an adequate diagnosis is essential for establishing better prevention strategies to avoid the circulation of abortion-related infectious agents in the herds.(AU)

Selection and characterization of peptides mimetic to Campylobacter fetus subsp. venerealis using phage display / Seleção e caracterização de peptídeos miméticos a Campylobacter fetus subsp. venerealis usando phage display

Bovine genital campylobacteriosis (BGC) is a venereal and subclinical disease that affects the fertility of cattle herds, and it is caused by Campylobacter fetus subsp. venerealis . This study selected peptides mimetic to the BGC-causing agent from a phage library. Phage display is a technique that applies bacteriophage libraries that reveal peptides fused to the viral capsid in biological selections against target proteins. Biopannings were performed for biological selection in the phage library using rabbit hyperimmune serum and C. fetus subsp. venerealis protein extract. Five selected heptapeptides were considered mimetic to Cfv-NCTC 10354 based on the results of bioinformatics analysis and assays with hyperimmune serum and cervicovaginal mucus obtained from heifers. ALASLPL and LSYLFPP were the most reactive peptides and considered promising as possible mimetic immunogens for C. fetus subsp. venerealis.

Campilobacteriose Genital Bovina (CGB) é uma doença venérea e subclínica que causa problemas reprodutivos em rebanhos, causada por Campylobacter fetus subsp. venerealis. Este trabalho teve como objetivo selecionar peptídeos miméticos ao agente da CGB de uma biblioteca de fagos. Phage display é uma técnica que aplica bibliotecas de bacteriófagos que expõem peptídeos fusionados ao capsídeo viral em seleções biológicas contra proteínas alvo. Biopannings foram realizados para seleção biológica na biblioteca de fagos por meio de soro hiperimune de coelho e extrato proteico de C. fetus subsp. venerealis. Cinco heptapeptídeos selecionados foram considerados miméticos para Cfv-NCTC 10354 a partir de análises de bioinformática e ensaios com soro hiperimune e muco cérvico-vaginal de novilhas. ALASLPL e LSYLFPP foram os peptídeos mais reativos e considerados promissores como possíveis imunógenos miméticos para C. fetus subsp. venerealis.

Selection and characterization of peptides mimetic to Campylobacter fetus subsp. venerealis using phage display / Seleção e caracterização de peptídeos miméticos a Campylobacter fetus subsp. venerealis usando phage display

Bovine genital campylobacteriosis (BGC) is a venereal and subclinical disease that affects the fertility of cattle herds, and it is caused by Campylobacter fetus subsp. venerealis . This study selected peptides mimetic to the BGC-causing agent from a phage library. Phage display is a technique that applies bacteriophage libraries that reveal peptides fused to the viral capsid in biological selections against target proteins. Biopannings were performed for biological selection in the phage library using rabbit hyperimmune serum and C. fetus subsp. venerealis protein extract. Five selected heptapeptides were considered mimetic to Cfv-NCTC 10354 based on the results of bioinformatics analysis and assays with hyperimmune serum and cervicovaginal mucus obtained from heifers. ALASLPL and LSYLFPP were the most reactive peptides and considered promising as possible mimetic immunogens for C. fetus subsp. venerealis.(AU)

Campilobacteriose Genital Bovina (CGB) é uma doença venérea e subclínica que causa problemas reprodutivos em rebanhos, causada por Campylobacter fetus subsp. venerealis. Este trabalho teve como objetivo selecionar peptídeos miméticos ao agente da CGB de uma biblioteca de fagos. Phage display é uma técnica que aplica bibliotecas de bacteriófagos que expõem peptídeos fusionados ao capsídeo viral em seleções biológicas contra proteínas alvo. Biopannings foram realizados para seleção biológica na biblioteca de fagos por meio de soro hiperimune de coelho e extrato proteico de C. fetus subsp. venerealis. Cinco heptapeptídeos selecionados foram considerados miméticos para Cfv-NCTC 10354 a partir de análises de bioinformática e ensaios com soro hiperimune e muco cérvico-vaginal de novilhas. ALASLPL e LSYLFPP foram os peptídeos mais reativos e considerados promissores como possíveis imunógenos miméticos para C. fetus subsp. venerealis.(AU)

Selection and characterization of peptides mimetic to Campylobacter fetus subsp. venerealis using phage display / Seleção e caracterização de peptídeos miméticos a Campylobacter fetus subsp. venerealis usando phage display

ABSTRACT: Bovine genital campylobacteriosis (BGC) is a venereal and subclinical disease that affects the fertility of cattle herds, and it is caused by Campylobacter fetus subsp. venerealis . This study selected peptides mimetic to the BGC-causing agent from a phage library. Phage display is a technique that applies bacteriophage libraries that reveal peptides fused to the viral capsid in biological selections against target proteins. Biopannings were performed for biological selection in the phage library using rabbit hyperimmune serum and C. fetus subsp. venerealis protein extract. Five selected heptapeptides were considered mimetic to Cfv-NCTC 10354 based on the results of bioinformatics analysis and assays with hyperimmune serum and cervicovaginal mucus obtained from heifers. ALASLPL and LSYLFPP were the most reactive peptides and considered promising as possible mimetic immunogens for C. fetus subsp. venerealis.

RESUMO: Campilobacteriose Genital Bovina (CGB) é uma doença venérea e subclínica que causa problemas reprodutivos em rebanhos, causada por Campylobacter fetus subsp. venerealis. Este trabalho teve como objetivo selecionar peptídeos miméticos ao agente da CGB de uma biblioteca de fagos. Phage display é uma técnica que aplica bibliotecas de bacteriófagos que expõem peptídeos fusionados ao capsídeo viral em seleções biológicas contra proteínas alvo. Biopannings foram realizados para seleção biológica na biblioteca de fagos por meio de soro hiperimune de coelho e extrato proteico de C. fetus subsp. venerealis. Cinco heptapeptídeos selecionados foram considerados miméticos para Cfv-NCTC 10354 a partir de análises de bioinformática e ensaios com soro hiperimune e muco cérvico-vaginal de novilhas. ALASLPL e LSYLFPP foram os peptídeos mais reativos e considerados promissores como possíveis imunógenos miméticos para C. fetus subsp. venerealis.

Chemotactic behavior of Campylobacter fetus subspecies towards cervical mucus, bovine placenta and selected substances and ion

Abstract The chemotaxis of C. fetus subsp. venerealis and C. fetus subsp. fetus was determined in the presence of bovine cervical mucus and bovine placental extract. Some reported substances and ion in those materials, such amino acids, ferrous iron, hormones, sugars and organic acids were also investigated. Bovine cervical mucus, bovine placenta extracts and some substances and ion of these materials namely L-fucose, L- aspartate, L-glutamate, L-serine, ferrous iron, fumarate, pyruvate and succinate were chemoattractants. The chemottraction was significantly larger in higher concentrations of the tested substances and ion and significant differences among tested strains were also observed. Meso-erythritol and hormones bovine placental lactogen, 17β-estradiol, and progesterone did not elicit chemotactical response. In conclusion, this chemotactic behavior may guide the C. fetus navigation in the bovine host's genital tract and be an important cofactor of tissue tropism for this bacterium.

Chemotactic behavior of Campylobacter fetus subspecies towards cervical mucus, bovine placenta and selected substances and ion

The chemotaxis of C. fetus subsp. venerealis and C. fetus subsp. fetus was determined in the presence of bovine cervical mucus and bovine placental extract. Some reported substances and ion in those materials, such amino acids, ferrous iron, hormones, sugars and organic acids were also investigated. Bovine cervical mucus, bovine placenta extracts and some substances and ion of these materials namely L–fucose, Laspartate, L–glutamate, L–serine, ferrous iron, fumarate, pyruvate and succinate were chemoattractants. The chemottraction was significantly larger in higher concentrations of the tested substances and ion and significant differences among tested strains were also observed. Meso-erythritol and hormones bovine placental lactogen, 17β-estradiol, and progesterone did not elicit chemotactical response. In conclusion, this chemotactic behavior may guide the C. fetus navigation in the bovine host's genital tract and be an important cofactor of tissue tropism for this bacterium.(AU)

Bovine genital campylobacteriosis: main features and perspectives for diagnosis and control / Campilobacteriose genital bovina: principais características e perspectivas para o diagnóstico e controle

Bovine genital campylobacteriosis (BGC) is a venereal disease caused by Campylobacter fetus subsp. venerealis. In countries with large cattle herds, such as Brazil, where the use of natural breeding as a reproductive strategy is a common practice, BGC is considered an important cause of reproductive failure and economic losses. In these cases, the bull is the asymptomatic carrier of the bacterium and the infected females can have infertility and even abortions. The techniques for the diagnosis of C. fetus are isolation in culture medium and identification by biochemical tests, immunofluorescence, immunoenzymatic assays and molecular techniques. Disease control is based on vaccination with bacterins. This review described the epidemiology, etiology, pathogenesis, and advances in the diagnosis and control of BGC.(AU)

A campilobacteriose genital bovina (CGB) é uma importante enfermidade de caráter venéreo causada por Campylobacter fetus subsp. venerealis. Em países com grandes rebanhos bovinos, como o Brasil, onde o uso da monta natural como estratégia reprodutiva é uma prática corrente, a CGB é considerada uma importante causa de falhas reprodutivas e perdas econômicas. Nestes casos, o touro é o portador assintomático da bactéria e as fêmeas infectadas podem apresentar infertilidade e até mesmo abortos. As técnicas para o diagnóstico de C. fetus são o isolamento em meio de cultura e identificação por testes bioquímicos; imunofluorescência; ensaios imunoenzimáticos e técnicas moleculares. O controle da doença é baseado em vacinação. Neste sentido, esta revisão consiste em uma abordagem sobre a epidemiologia, a etiologia, a patogenia, os avanços no diagnóstico e controle da CGB.(AU)

Causes of abortion in dairy cows in Uruguay / Causas de aborto em bovinos de leite no Uruguai

A case series study was conducted to determine the frequency of causes of abortion in dairy cattle in Uruguay. The sample size of 102 cases was composed of 53 fetuses, 35 fetuses with placentas, and 14 placentas without an associated fetus. All cases underwent gross and microscopic pathologic examinations as well as microbiological and serological testing. The etiology was determined in 54 (53%) of cases, 51 of which were caused by infectious agents. Within the observed 102 cases, 30 (29%) were caused by Neospora caninum, six (6%) by Coxiella burnetii and two (2%) by Campylobacter fetus subsp. venerealis. Bovine Parainfluenza-3 virus and Salmonella enterica serovar Newport caused one abortion each. Opportunistic bacteria (Escherichia coli, Streptococcus sp., Staphylococcus sp., Mannheimia sp., Trueperella pyogenes, and Providencia stuartii) were associated with 11 abortions. In two cases the fetal death was attributed to dystocia, and in one case the fetus had a congenital mesothelioma. Bovine viral diarrhea virus (BVDV) infection was identified in three fetuses; two of which were co-infected with and had typical lesions of N. caninum. No lesions were observed in the other fetus infected by BVDV. Leptospira interrogans was identified in one fetus without lesions. Despite the relatively low overall success rate in establishing an etiological diagnosis in cases of abortion in cattle, a systemic workup of bovine abortion is necessary to establish prevention and control strategies. This also facilitates monitoring and surveillance of reproductive diseases in dairy cattle, some of which represent a risk to public health.(AU)

Uma série de casos foi estudada para determinar a frequência de causas do aborto em bovinos leiteiros no Uruguai. A amostra, de 102 casos, foi composta por 53 fetos, 35 fetos com placentas e 14 placentas sem feto associado. Todos os casos foram submetidos a exames patológicos macroscópicos e microscópicos, além de testes microbiológicos e sorológicos. A etiologia foi determinada em 54 (53%) dos casos, 51 dos quais foram causados por agentes infecciosos. Nos 102 casos observados, 30 (29%) foram causados por Neospora caninum, seis (6%) por Coxiella burnetii e dois (2%) por Campylobacter fetus subsp. venerealis. O vírus da Parainfluenza-3 e Salmonella enterica serovar Newport causaram um aborto cada. Bactérias oportunistas (Escherichia coli, Streptococcus sp., Staphylococcus sp., Mannheimia sp., Trueperella pyogenes e Providencia stuartii) foram associadas a 11 abortos. Em dois casos, a morte fetal foi atribuída a distocia e, em um caso, o feto apresentava mesotelioma congênito. A infecção pelo vírus da diarreia viral bovina (BVDV) foi identificada em três fetos; dois dos quais foram co-infectados e apresentavam lesões típicas de N. caninum. Não foram observadas lesões no outro feto infectado pelo BVDV. Leptospira interrogans foi identificada em um feto sem lesões. Apesar da relativamente baixa taxa de sucesso no diagnóstico etiológico nos casos de aborto em bovinos, é necessário o diagnóstico sistemático dos abortos para estabelecer estratégias de prevenção e controle. Isso também facilita o monitoramento e a vigilância de doenças reprodutivas em bovinos leiteiros, algumas das quais representam um risco para a saúde pública.(AU)

Causes of abortion in dairy cows in Uruguay / Causas de aborto em bovinos de leite no Uruguai

A case series study was conducted to determine the frequency of causes of abortion in dairy cattle in Uruguay. The sample size of 102 cases was composed of 53 fetuses, 35 fetuses with placentas, and 14 placentas without an associated fetus. All cases underwent gross and microscopic pathologic examinations as well as microbiological and serological testing. The etiology was determined in 54 (53%) of cases, 51 of which were caused by infectious agents. Within the observed 102 cases, 30 (29%) were caused by Neospora caninum, six (6%) by Coxiella burnetii and two (2%) by Campylobacter fetus subsp. venerealis. Bovine Parainfluenza-3 virus and Salmonella enterica serovar Newport caused one abortion each. Opportunistic bacteria (Escherichia coli, Streptococcus sp., Staphylococcus sp., Mannheimia sp., Trueperella pyogenes, and Providencia stuartii) were associated with 11 abortions. In two cases the fetal death was attributed to dystocia, and in one case the fetus had a congenital mesothelioma. Bovine viral diarrhea virus (BVDV) infection was identified in three fetuses; two of which were co-infected with and had typical lesions of N. caninum. No lesions were observed in the other fetus infected by BVDV. Leptospira interrogans was identified in one fetus without lesions. Despite the relatively low overall success rate in establishing an etiological diagnosis in cases of abortion in cattle, a systemic workup of bovine abortion is necessary to establish prevention and control strategies. This also facilitates monitoring and surveillance of reproductive diseases in dairy cattle, some of which represent a risk to public health.(AU)

Uma série de casos foi estudada para determinar a frequência de causas do aborto em bovinos leiteiros no Uruguai. A amostra, de 102 casos, foi composta por 53 fetos, 35 fetos com placentas e 14 placentas sem feto associado. Todos os casos foram submetidos a exames patológicos macroscópicos e microscópicos, além de testes microbiológicos e sorológicos. A etiologia foi determinada em 54 (53%) dos casos, 51 dos quais foram causados por agentes infecciosos. Nos 102 casos observados, 30 (29%) foram causados por Neospora caninum, seis (6%) por Coxiella burnetii e dois (2%) por Campylobacter fetus subsp. venerealis. O vírus da Parainfluenza-3 e Salmonella enterica serovar Newport causaram um aborto cada. Bactérias oportunistas (Escherichia coli, Streptococcus sp., Staphylococcus sp., Mannheimia sp., Trueperella pyogenes e Providencia stuartii) foram associadas a 11 abortos. Em dois casos, a morte fetal foi atribuída a distocia e, em um caso, o feto apresentava mesotelioma congênito. A infecção pelo vírus da diarreia viral bovina (BVDV) foi identificada em três fetos; dois dos quais foram co-infectados e apresentavam lesões típicas de N. caninum. Não foram observadas lesões no outro feto infectado pelo BVDV. Leptospira interrogans foi identificada em um feto sem lesões. Apesar da relativamente baixa taxa de sucesso no diagnóstico etiológico nos casos de aborto em bovinos, é necessário o diagnóstico sistemático dos abortos para estabelecer estratégias de prevenção e controle. Isso também facilita o monitoramento e a vigilância de doenças reprodutivas em bovinos leiteiros, algumas das quais representam um risco para a saúde pública.(AU)

Antimicrobial Resistance in Campylobacter jejuni Isolated from Brazilian Poultry Slaughterhouses

Campylobacteriosis is one of the most common foodborne diseases in the world. It is considered the most frequently reported foodborne illness in the European Union (EU) and one of the most important in the United States (US) (EFSA & ECDC, 2018; CDC, 2019a; WHO, 2019). Poultry is known to be the major reservoir and an important source for pathogen transmission to humans (Kaakoush et al., 2015). Campylobacteriosis is most often associated with the consumption of raw and undercooked poultry or the cross-contamination of other foods by these items (CDC, 2019a). Although Brazil is a leading supplier of the worlds poultry meat (ABPA, 2018), Brazils official data does not report Campylobacter infections.(AU)

Antimicrobial Resistance in Campylobacter jejuni Isolated from Brazilian Poultry Slaughterhouses

Campylobacteriosis is one of the most common foodborne diseases in the world. It is considered the most frequently reported foodborne illness in the European Union (EU) and one of the most important in the United States (US) (EFSA & ECDC, 2018; CDC, 2019a; WHO, 2019). Poultry is known to be the major reservoir and an important source for pathogen transmission to humans (Kaakoush et al., 2015). Campylobacteriosis is most often associated with the consumption of raw and undercooked poultry or the cross-contamination of other foods by these items (CDC, 2019a). Although Brazil is a leading supplier of the world’s poultry meat (ABPA, 2018), Brazil’s official data does not report Campylobacter infections.

Phenotypic and molecular characterization of erythromycin resistance in Campylobacter jejuni and Campylobacter coli strains isolated from swine and broiler chickens / Caracterização fenotípica e molecular da resistência à eritromicina em cepas de Campylobacter jejuni e Campylobacter coli isoladas de suínos e frangos de corte

Campylobacter spp. is a bacterial agent that causes gastroenteritis in humans and may trigger Guillain-Barré Syndrome (GBS) and is also considered one of the main foodborne diseases in developed countries. Poultry and pigs are considered reservoirs of these microorganisms, as well as raw or undercooked by-products are often incriminated as a source of human infection. Treatment in human cases is with macrolide, such erythromycin, that inhibits the protein synthesis of the microorganism. This study aimed to isolate Campylobacter jejuni and Campylobacter coli from intestinal content samples of broiler chickens (n=20) and swine (n=30) to characterize the erythromycin resistance profile of the strains and to detect molecular mechanisms involved in this resistance. The minimum inhibitory concentration was determined by agar dilution. The Mismatch Amplification Mutation Assay-Polymerase Chain Reaction (MAMA-PCR) was performed to detect mutations at positions 2074 and 2075 of 23S rRNA region, in addition to PCR test to detect the erm(B) gene. From the intestinal content of broiler chickens, 18 strains of C. jejuni and two strains of C. coli were isolated, whereas, from swine samples, no C. jejuni strain and 14 strains of C. coli were isolated. All C. coli strains were resistant, and three C. jejuni strains from broilers chickens were characterized with intermediate resistance to erythromycin. The MIC of the strains ranged from ≤0.5mg/μL to ≥128mg/μL. All resistant strains had the A2075G mutation, and one strain with intermediate resistance had the A2075G mutation. However, the A2074C mutation and the erm(B) gene were not detected. High resistance levels were detected in C. coli strains isolated from swine. The MAMA-PCR is a practical tool for detecting the erythromycin resistance in Campylobacter strains.(AU)

Campylobacter spp. é um agente bacteriano causador de gastroenterite em humanos e associado à síndrome de Guillain-Barré, sendo a campilobacteriose considerada uma das principais enfermidades de origem alimentar. Aves e suínos são importantes reservatórios desses microrganismos e seus produtos derivados crus ou mal cozidos são muitas vezes incriminados como fonte de infecção humana. A primeira escolha para o tratamento em casos humanos são os antimicrobianos da classe dos macrolídeos como à eritromicina. Dentro desse contexto, o objetivo deste estudo foi isolar Campylobacter jejuni e C. coli a partir de 20 amostras de conteúdo intestinal de frangos de corte e de 30 de suínos ao abate e investigar a resistência à eritromicina das estirpes obtidas e os possíveis mecanismos moleculares envolvidos nesta resistência. A concentração inibitória mínima foi determinada pela diluição em ágar e a técnica MAMA-PCR foi utilizada para detecção de mutações nas posições 2074 e 2075 da região 23s rRNA, foi pesquisado também a presença do gene erm(B) pela PCR. A partir do conteúdo intestinal de frangos de corte foram isoladas 18 estirpes de C. jejuni e duas de C. coli, enquanto de suínos foram obtidas 14 estirpes de C. coli e nenhuma estirpe de C. jejuni. Todas as estirpes de C. coli de suínos foram identificadas como resistentes e três estirpes de C. jejuni de frangos foram caracterizadas com resistência intermediária. A CIM das estirpes variou de ≤0,5mg/μL a ≥128mg/μL. Todas as estirpes resistentes tinham a mutação A2075G e uma cepa com resistência intermediária também apresentou a mutação A2075G. Não foi detectada a mutação A2074C ou a presença do gene erm(B) em nenhuma das estirpes obtidas. Os resultados revelam um alto nível de resistência em estirpes de C. coli isoladas de suínos frente a eritromicina. A técnica MAMA PCR utilizada se constitui em uma ferramenta prática para detecção da resistência à eritromicina em estirpes de C. jejuni e C. coli.(AU)

Phenotypic and molecular characterization of erythromycin resistance in Campylobacter jejuni and Campylobacter coli strains isolated from swine and broiler chickens / Caracterização fenotípica e molecular da resistência à eritromicina em cepas de Campylobacter jejuni e Campylobacter coli isoladas de suínos e frangos de corte

Campylobacter spp. is a bacterial agent that causes gastroenteritis in humans and may trigger Guillain-Barré Syndrome (GBS) and is also considered one of the main foodborne diseases in developed countries. Poultry and pigs are considered reservoirs of these microorganisms, as well as raw or undercooked by-products are often incriminated as a source of human infection. Treatment in human cases is with macrolide, such erythromycin, that inhibits the protein synthesis of the microorganism. This study aimed to isolate Campylobacter jejuni and Campylobacter coli from intestinal content samples of broiler chickens (n=20) and swine (n=30) to characterize the erythromycin resistance profile of the strains and to detect molecular mechanisms involved in this resistance. The minimum inhibitory concentration was determined by agar dilution. The Mismatch Amplification Mutation Assay-Polymerase Chain Reaction (MAMA-PCR) was performed to detect mutations at positions 2074 and 2075 of 23S rRNA region, in addition to PCR test to detect the erm(B) gene. From the intestinal content of broiler chickens, 18 strains of C. jejuni and two strains of C. coli were isolated, whereas, from swine samples, no C. jejuni strain and 14 strains of C. coli were isolated. All C. coli strains were resistant, and three C. jejuni strains from broilers chickens were characterized with intermediate resistance to erythromycin. The MIC of the strains ranged from ≤0.5mg/μL to ≥128mg/μL. All resistant strains had the A2075G mutation, and one strain with intermediate resistance had the A2075G mutation. However, the A2074C mutation and the erm(B) gene were not detected. High resistance levels were detected in C. coli strains isolated from swine. The MAMA-PCR is a practical tool for detecting the erythromycin resistance in Campylobacter strains.(AU)

Campylobacter spp. é um agente bacteriano causador de gastroenterite em humanos e associado à síndrome de Guillain-Barré, sendo a campilobacteriose considerada uma das principais enfermidades de origem alimentar. Aves e suínos são importantes reservatórios desses microrganismos e seus produtos derivados crus ou mal cozidos são muitas vezes incriminados como fonte de infecção humana. A primeira escolha para o tratamento em casos humanos são os antimicrobianos da classe dos macrolídeos como à eritromicina. Dentro desse contexto, o objetivo deste estudo foi isolar Campylobacter jejuni e C. coli a partir de 20 amostras de conteúdo intestinal de frangos de corte e de 30 de suínos ao abate e investigar a resistência à eritromicina das estirpes obtidas e os possíveis mecanismos moleculares envolvidos nesta resistência. A concentração inibitória mínima foi determinada pela diluição em ágar e a técnica MAMA-PCR foi utilizada para detecção de mutações nas posições 2074 e 2075 da região 23s rRNA, foi pesquisado também a presença do gene erm(B) pela PCR. A partir do conteúdo intestinal de frangos de corte foram isoladas 18 estirpes de C. jejuni e duas de C. coli, enquanto de suínos foram obtidas 14 estirpes de C. coli e nenhuma estirpe de C. jejuni. Todas as estirpes de C. coli de suínos foram identificadas como resistentes e três estirpes de C. jejuni de frangos foram caracterizadas com resistência intermediária. A CIM das estirpes variou de ≤0,5mg/μL a ≥128mg/μL. Todas as estirpes resistentes tinham a mutação A2075G e uma cepa com resistência intermediária também apresentou a mutação A2075G. Não foi detectada a mutação A2074C ou a presença do gene erm(B) em nenhuma das estirpes obtidas. Os resultados revelam um alto nível de resistência em estirpes de C. coli isoladas de suínos frente a eritromicina. A técnica MAMA PCR utilizada se constitui em uma ferramenta prática para detecção da resistência à eritromicina em estirpes de C. jejuni e C. coli.(AU)

Maintenance of strains of Campylobacter jejuni in laboratories after use of cryoprotectors and pre-treatment of stress / Manutenção de cepas de Campylobacter jejuni em laboratório após uso de crioprotetores e pré-tratamento de estresse

This study evaluated the feasibility and the production of transcripts of sodB, p19, ciaB and dnaJ genes in strains of Campylobacter jejuni ATCC 33291, NCTC 11351, and 2383 IAL stored in whole UHT milk and or neopepton + 12% glycerol, submitted or not to pre-treatments at 4°C or 10°C for 30 minutes. The analyzes were performed immediately after freezing in liquid nitrogen (day 0) and after maintenance for 30, 60, and 90 days at -20ºC. The viability was evaluated by the traditional culture method and the production of transcripts by the RT-PCR technique. The quantification was only possible on the first day of analysis (day 0) and presented a mean of 3.0 x 107 CFU, and in the other periods of storage the strains presented confluent growth, not allowing their enumeration. The results indicated that whole UHT milk was more adequate for cryopreservation than the use of neopepton + 12% glycerol. The use of pre-treatments combined with the use of UHT milk as a cryoprotective medium stabilized the cells in order to transcribe the ciaB, dnaJ, sodBand p19 genes in the strains maintained under -20 ° for 30 to 60 days, indicating that they are more suitable methods for the maintenance of strains in the laboratory.

Este estudo avaliou a viabilidade e a produção de transcritos dos genes sodB, p19, ciaB e dnaJ em cepas de Campylobacter jejuni ATCC 33291, NCTC 11351 e 2383 IAL armazenadas em leite integral UHT e / ou neopeptona + 12% glicerol, submetidas ou não a pré-tratamentos a 4°C ou 10°C durante 30 minutos. As análises foram realizadas imediatamente após o congelamento em nitrogênio líquido (dia 0) e após a manutenção por 30, 60 e 90 dias à -20ºC. A viabilidade foi avaliada pelo método tradicional de cultura e a produção de transcritos, pela técnica de RT-PCR. A quantificação só foi possível no primeiro dia de análise (dia 0) e apresentou uma média de 3,0 x 107 UFC, e nos demais períodos de armazenamento as cepas apresentaram crescimento confluente, não permitindo sua enumeração. Os resultados indicaram que o leite integral UHT foi mais adequado para criopreservação em relação ao uso de neopeptona + 12% glicerol. A utilização de pré-tratamentos combinados com o uso de leite UHT como meio crioprotetor estabilizou as células para transcrever os genes ciaB, dnaJ, sodB e p19 nas cepas mantidas abaixo de -20° por 30 a 60 dias, indicando que são métodos mais adequados para a manutenção de cepas em laboratório.

Maintenance of strains of Campylobacter jejuni in laboratories after use of cryoprotectors and pre-treatment of stress / Manutenção de cepas de Campylobacter jejuni em laboratório após uso de crioprotetores e pré-tratamento de estresse

This study evaluated the feasibility and the production of transcripts of sodB, p19, ciaB and dnaJ genes in strains of Campylobacter jejuni ATCC 33291, NCTC 11351, and 2383 IAL stored in whole UHT milk and or neopepton + 12% glycerol, submitted or not to pre-treatments at 4°C or 10°C for 30 minutes. The analyzes were performed immediately after freezing in liquid nitrogen (day 0) and after maintenance for 30, 60, and 90 days at -20ºC. The viability was evaluated by the traditional culture method and the production of transcripts by the RT-PCR technique. The quantification was only possible on the first day of analysis (day 0) and presented a mean of 3.0 x 107 CFU, and in the other periods of storage the strains presented confluent growth, not allowing their enumeration. The results indicated that whole UHT milk was more adequate for cryopreservation than the use of neopepton + 12% glycerol. The use of pre-treatments combined with the use of UHT milk as a cryoprotective medium stabilized the cells in order to transcribe the ciaB, dnaJ, sodBand p19 genes in the strains maintained under -20 ° for 30 to 60 days, indicating that they are more suitable methods for the maintenance of strains in the laboratory.(AU)

Este estudo avaliou a viabilidade e a produção de transcritos dos genes sodB, p19, ciaB e dnaJ em cepas de Campylobacter jejuni ATCC 33291, NCTC 11351 e 2383 IAL armazenadas em leite integral UHT e / ou neopeptona + 12% glicerol, submetidas ou não a pré-tratamentos a 4°C ou 10°C durante 30 minutos. As análises foram realizadas imediatamente após o congelamento em nitrogênio líquido (dia 0) e após a manutenção por 30, 60 e 90 dias à -20ºC. A viabilidade foi avaliada pelo método tradicional de cultura e a produção de transcritos, pela técnica de RT-PCR. A quantificação só foi possível no primeiro dia de análise (dia 0) e apresentou uma média de 3,0 x 107 UFC, e nos demais períodos de armazenamento as cepas apresentaram crescimento confluente, não permitindo sua enumeração. Os resultados indicaram que o leite integral UHT foi mais adequado para criopreservação em relação ao uso de neopeptona + 12% glicerol. A utilização de pré-tratamentos combinados com o uso de leite UHT como meio crioprotetor estabilizou as células para transcrever os genes ciaB, dnaJ, sodB e p19 nas cepas mantidas abaixo de -20° por 30 a 60 dias, indicando que são métodos mais adequados para a manutenção de cepas em laboratório.(AU)

Detecção de Campylobacter jejuni em produtos de frango utilizando separação imunomagnética / Detection of Campylobacter jejuni in chicken products using immunomagnetic separation

Campylobacter jejuni é o principal causador de gastroenterite bacteriana aguda, e a carne de frango tem se mostrado uma importante fonte de transmissão. Este microrganismo é de difícil isolamento e os métodos convencionais muitas vezes não são eficientes, podendo levar a resultados errôneos. O objetivo deste trabalho foi desenvolver e testar a técnica de separação imunomagnética (IMS) na detecção de C. jejuni em produtos de frango. Micropartículas magnéticas ligadas a anticorpos policlonais anti-C. jejuni foram utilizadas para concentrar C. jejuni antes da semeadura em ágar. O protocolo foi comparado com o método convencional. C. jejuni foi recuperado do alimento experimentalmente contaminado por ambos os métodos, entretanto, quando foi usada a IMS, a presença de microrganismos contaminantes nos meios de cultura foi menor. C. jejuni foi isolado de 7% das amostras de alimento naturalmente contaminadas, usando IMS, e de 3% pelo método convencional. C. coli foi isolado de uma amostra pelo método convencional, mas não foi detectado pelo protocolo com IMS. A técnica de IMS pode ser usada para isolamento de C. jejuni de alimentos, oferecendo a vantagem de detectar em amostras o microrganismo cujo isolamento não é obtido por meio do método convencional.(AU)

Campylobacter jejuni is the main cause of acute bacterial gastroenteritis and chicken meat has shown to be an important source of infection. This microorganism is difficult to isolate and the conventional methods are often inefficient and may lead to erroneous results. This study aimed at developing and testing the technique of immunomagnetic separation (IMS) in the detection of C. jejuni in chicken products. Microparticles magnetically connected anti-C. jejuni polyclonal antibodies were used to concentrate C. jejuni before agar seeding. The protocol was compared with the conventional method. C. jejuni was recovered from experimentally contaminated food for both methods, however, when the IMS was used, the presence of contaminating microorganisms in the means of culture was smaller. C. jejuni was isolated from 7% of samples of food naturally contaminated, using IMS, and 3% by conventional method. C. coli was isolated from a sample by conventional method, but it was not detected by protocol with IMS. The IMS technique can be used for isolation of C. jejuni in food, offering the advantage of detecting the microorganism in samples from which the isolation is not obtained with the use of the conventional method.(AU)

Detecção de Campylobacter jejuni em produtos de frango utilizando separação imunomagnética / Detection of Campylobacter jejuni in chicken products using immunomagnetic separation

Campylobacter jejuni é o principal causador de gastroenterite bacteriana aguda, e a carne de frango tem se mostrado uma importante fonte de transmissão. Este microrganismo é de difícil isolamento e os métodos convencionais muitas vezes não são eficientes, podendo levar a resultados errôneos. O objetivo deste trabalho foi desenvolver e testar a técnica de separação imunomagnética (IMS) na detecção de C. jejuni em produtos de frango. Micropartículas magnéticas ligadas a anticorpos policlonais anti-C. jejuni foram utilizadas para concentrar C. jejuni antes da semeadura em ágar. O protocolo foi comparado com o método convencional. C. jejuni foi recuperado do alimento experimentalmente contaminado por ambos os métodos, entretanto, quando foi usada a IMS, a presença de microrganismos contaminantes nos meios de cultura foi menor. C. jejuni foi isolado de 7% das amostras de alimento naturalmente contaminadas, usando IMS, e de 3% pelo método convencional. C. coli foi isolado de uma amostra pelo método convencional, mas não foi detectado pelo protocolo com IMS. A técnica de IMS pode ser usada para isolamento de C. jejuni de alimentos, oferecendo a vantagem de detectar em amostras o microrganismo cujo isolamento não é obtido por meio do método convencional.(AU)

Campylobacter jejuni is the main cause of acute bacterial gastroenteritis and chicken meat has shown to be an important source of infection. This microorganism is difficult to isolate and the conventional methods are often inefficient and may lead to erroneous results. This study aimed at developing and testing the technique of immunomagnetic separation (IMS) in the detection of C. jejuni in chicken products. Microparticles magnetically connected anti-C. jejuni polyclonal antibodies were used to concentrate C. jejuni before agar seeding. The protocol was compared with the conventional method. C. jejuni was recovered from experimentally contaminated food for both methods, however, when the IMS was used, the presence of contaminating microorganisms in the means of culture was smaller. C. jejuni was isolated from 7% of samples of food naturally contaminated, using IMS, and 3% by conventional method. C. coli was isolated from a sample by conventional method, but it was not detected by protocol with IMS. The IMS technique can be used for isolation of C. jejuni in food, offering the advantage of detecting the microorganism in samples from which the isolation is not obtained with the use of the conventional method.(AU)

Isolation and antimicrobial resistance of Campylobacter jejuni and Campylobacter coli found in chilled chicken carcasses in the Federal District Region and surrounding areas / Isolamento e resistência antimicrobiana de Campylobacter jejuni e Campylobacter coli encontradas em carcaças de frango resfriadas na região do Distrito Federal e entorno

This study aimed to isolate Campylobacter jejuni and Campylobacter coli from chilled chicken carcasses marketed in the Federal District Region and surrounding areas, as well as to detect the occurrence of antimicrobial resistance and genes responsible for the same. A total of 105 chilled chicken carcasses were collected, of which 7 (6.67%) were positive for C. jejuni and 4 (3.81%) were positive for C. coli. These results were obtained using both the conventional microbiological isolation method and polymerase chain reaction assays. All of the positive strains were subjected to antimicrobial susceptibility testing for seven antimicrobials. The resistance incidences found in the C. jejuni strains were as follows: 71.43% for tetracycline and nalidixic acid, 42.86% for streptomycin and gentamicin, 57.14% for ciprofloxacin and erythromycin, and 28.57% for chloramphenicol. Among the C. coli strains, 100% were resistant to tetracycline and streptomycin, 75% were resistant to erythromycin, 50% were resistant to ciprofloxacin, gentamicin, and nalidixic acid, and no strains were resistant to chloramphenicol. While analyzing the presence of antimicrobial resistance genes in the isolated C. jejuni strains, the aph3-1 (resistance to aminoglycosides), aadE (resistance to streptomycin), and tet(O) (resistance to tetracycline) genes were identified, with occurrence rates of 57.14%, 28.57%, and 42.86%, respectively, whereas in the C. coli strains, there was a 25% occurrence rate for both the aph3-1 and tet(O) genes. The aadE gene was not found in the C. coli isolates. The results of this study demonstrated the presence of C. jejuni and C. coli in chilled chicken carcasses marketed in the Federal District Region and surrounding areas, as well as the antimicrobial resistance and the presence of resistance genes in these bacteria, which may pose threats to public health.

O objetivo deste trabalho foi isolar Campylobacter jejuni e Campylobacter coli de carcaças de frango resfriadas comercializadas no Distrito Federal e entorno, bem como detectar a ocorrência de resistência antimicrobiana e genes responsáveis pela resistência antimicrobiana. Foram coletadas um total de 105 carcaças de frango resfriadas, das quais 7 (6,67%) foram positivas para C. jejuni e 4 (3,81%) para C. coli. Estes resultados foram obtidos usando tanto o método convencional de isolamento microbiológico quanto os ensaios de reação em cadeia da polymerase (PCR). Todas as cepas positivas foram submetidas ao teste de susceptibilidade antimicrobiana para sete antimicrobianos. As incidências de resistência encontradas nas cepas de C. jejuni foram as seguintes: 71,43% para tetraciclina e ácido nalidíxico, 42,86% para estreptomicina e gentamicina, 57,14% para ciprofloxacina e eritromicina e 28,57% para cloranfenicol. Entre as cepas de C. coli, 100% foram resistentes à tetraciclina e à estreptomicina, 75% eram resistentes à eritromicina, 50% eram resistentes à ciprofloxacina, gentamicina e ácido nalidíxico, e nenhuma cepa eram resistentes ao cloranfenicol. Ao analisar a presença de genes de resistência antimicrobiana nas cepas isoladas de C. jejuni, foram identificados os genes aph3-1 (resistência a aminoglicosídeos), aadE (resistência à estreptomicina) e tet (O) (resistência à tetraciclina) genes foram identificados, com taxas de ocorrência de 57,14%, 28,57% e 42,86%, respectivamente, enquanto que nas cepas de C. coli, houve uma taxa de ocorrência de 25% para os genes aph3-1 e tet (O). O gene aadE não foi encontrado nos isolados de C. coli. Os resultados deste estudo demonstraram a presença de C. jejuni e C. coli em carcaças de frango resfriadas comercializadas na Região do Distrito Federal e entorno, além da resistência antimicrobiana e a presença de genes de resistência nestas cepas, que podem se tornar uma possível ameaça a saúde pública.

Isolation and antimicrobial resistance of Campylobacter jejuni and Campylobacter coli found in chilled chicken carcasses in the Federal District Region and surrounding areas / Isolamento e resistência antimicrobiana de Campylobacter jejuni e Campylobacter coli encontradas em carcaças de frango resfriadas na região do Distrito Federal e entorno

This study aimed to isolate Campylobacter jejuni and Campylobacter coli from chilled chicken carcasses marketed in the Federal District Region and surrounding areas, as well as to detect the occurrence of antimicrobial resistance and genes responsible for the same. A total of 105 chilled chicken carcasses were collected, of which 7 (6.67%) were positive for C. jejuni and 4 (3.81%) were positive for C. coli. These results were obtained using both the conventional microbiological isolation method and polymerase chain reaction assays. All of the positive strains were subjected to antimicrobial susceptibility testing for seven antimicrobials. The resistance incidences found in the C. jejuni strains were as follows: 71.43% for tetracycline and nalidixic acid, 42.86% for streptomycin and gentamicin, 57.14% for ciprofloxacin and erythromycin, and 28.57% for chloramphenicol. Among the C. coli strains, 100% were resistant to tetracycline and streptomycin, 75% were resistant to erythromycin, 50% were resistant to ciprofloxacin, gentamicin, and nalidixic acid, and no strains were resistant to chloramphenicol. While analyzing the presence of antimicrobial resistance genes in the isolated C. jejuni strains, the aph3-1 (resistance to aminoglycosides), aadE (resistance to streptomycin), and tet(O) (resistance to tetracycline) genes were identified, with occurrence rates of 57.14%, 28.57%, and 42.86%, respectively, whereas in the C. coli strains, there was a 25% occurrence rate for both the aph3-1 and tet(O) genes. The aadE gene was not found in the C. coli isolates. The results of this study demonstrated the presence of C. jejuni and C. coli in chilled chicken carcasses marketed in the Federal District Region and surrounding areas, as well as the antimicrobial resistance and the presence of resistance genes in these bacteria, which may pose threats to public health.(AU)

O objetivo deste trabalho foi isolar Campylobacter jejuni e Campylobacter coli de carcaças de frango resfriadas comercializadas no Distrito Federal e entorno, bem como detectar a ocorrência de resistência antimicrobiana e genes responsáveis pela resistência antimicrobiana. Foram coletadas um total de 105 carcaças de frango resfriadas, das quais 7 (6,67%) foram positivas para C. jejuni e 4 (3,81%) para C. coli. Estes resultados foram obtidos usando tanto o método convencional de isolamento microbiológico quanto os ensaios de reação em cadeia da polymerase (PCR). Todas as cepas positivas foram submetidas ao teste de susceptibilidade antimicrobiana para sete antimicrobianos. As incidências de resistência encontradas nas cepas de C. jejuni foram as seguintes: 71,43% para tetraciclina e ácido nalidíxico, 42,86% para estreptomicina e gentamicina, 57,14% para ciprofloxacina e eritromicina e 28,57% para cloranfenicol. Entre as cepas de C. coli, 100% foram resistentes à tetraciclina e à estreptomicina, 75% eram resistentes à eritromicina, 50% eram resistentes à ciprofloxacina, gentamicina e ácido nalidíxico, e nenhuma cepa eram resistentes ao cloranfenicol. Ao analisar a presença de genes de resistência antimicrobiana nas cepas isoladas de C. jejuni, foram identificados os genes aph3-1 (resistência a aminoglicosídeos), aadE (resistência à estreptomicina) e tet (O) (resistência à tetraciclina) genes foram identificados, com taxas de ocorrência de 57,14%, 28,57% e 42,86%, respectivamente, enquanto que nas cepas de C. coli, houve uma taxa de ocorrência de 25% para os genes aph3-1 e tet (O). O gene aadE não foi encontrado nos isolados de C. coli. Os resultados deste estudo demonstraram a presença de C. jejuni e C. coli em carcaças de frango resfriadas comercializadas na Região do Distrito Federal e entorno, além da resistência antimicrobiana e a presença de genes de resistência nestas cepas, que podem se tornar uma possível ameaça a saúde pública.(AU)