Fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli from poultry and human samples assessed by PCR-restriction fragment length polymorphism assay

The objective of this study was to determine fluoroquinolone resistance in Campylobacter spp from poultry and human isolates. Forty-one Campylobacter jejuni isolates (30 of poultry origin and 11 of human origin) and 11 Campylobacter coli isolates (10 of human origin and 1 of poultry origin) were examined for ciprofloxacin, norfloxacin, and nalidixic acid resistance using the minimal inhibitory concentration (MIC) method. Thereafter, the isolates were analyzed by PCR±Restriction Fragment Length Polymorphism (RFLP) assay for detection of Thr-86 mutation. Finally, DNA sequencing was performed for confirmation of gyrA gene mutation. A complete correlation was observed between MICs, PCR-RFLP assay, and sequencing. The results revealed high quinolone resistance rates for C. jejuni (100%) and C. coli (100%) isolates obtained from poultry and moderate resistance for C. jejuni (9.1%) and C. coli (40%) samples of human origin. A mutation in codon 86 of the gyrA gene with a Thr-to-Ile substitution is reported to be the main cause of high resistance to quinolones. This mutation can be analyzed by PCR-RFLP assay, which has been proven to be a simple and fast method for the detection of fluoroquinolone resistance in Campylobacter spp

Identification of pathogenic genes in Campylobacter jejuni isolated from broiler carcasses and broiler slaughterhouses

Campylobacter jejuni is one of the most common causes of foodborne diseases worldwide. There are few reports on Campylobacter strains isolated from Latin-American countries. Here, 140 C. jejuni strains isolated from cloacal and transport boxes swabs, water from chiller tanks, and broiler carcasses of five poultry companies in Southern Brazil were identified using phenotypic and genotypic methods. Polymerase chain reaction (PCR) was used to analyze eight C. jejuni virulence markers: flaA, cadF, and invasion-associated (iam) genes, cdtABC operon (associated with the cytolethal distending toxin), and plasmidial virB11 and wlaN genes were present in 78.5%, 77.8%, 0%, 74.2%, 22.1%, and 10.7% of samples, respectively. There were 25 different virulence profiles: 1 (cdtA, cdtB, cdtC, flaA, and cadF), 2 (cdtA, cdtB, cdtC, flaA, cadF, and virB11), and 3 (cdtA, cdtB, cdtC, flaA, cadF, and wlaN) were the most common (> 60% of strains). We provide insight into factors related to the occurrence of this pathogen and their epidemiology

Comparison of transport crates contamination with Campylobacter spp. before and after the cleaning and disinfection procedure in broiler slaughterhouses

Campylobacteriosis is one of the most common types of bacterial gastroenteritis affecting humans, and poultry is considered a major source of the causative organism, Campylobacter spp. Broilers may arrive contaminated at slaughterhouses, and transport crates could be considered a potential source of contamination. Thus, cleaning and disinfection procedures are crucial to avoid cross-contamination among flocks. Despite its public health importance in Latin American countries, virulence factors of Campylobacter jejuni remain poorly studied in this region. Thus, this study aimed to: 1) determine the occurrence of contaminated crates at a poultry slaughterhouse, 2) compare the contamination before and after the cleaning and disinfection procedures, and 3) detect virulence-associated genes in C. jejuni strains by PCR. Campylobacter spp. were recovered from 8 of the 10 flocks evaluated, and C. jejuni was detected as the main species. There was no significant difference in the Campylobacter detection or quantification between crates at the reception platform and crates after the cleaning/disinfection processes. However, crates after 24 h of natural drying, presented a significant (P 0.05) in the detection of two C. jejuni virulence genes, flaA (encode a major flagellin protein) and cadF (encode an adhesion and fibronectin-binding protein), among various stages of the cleaning and disinfection processes. Our results demonstrate the high contamination levels of Campylobacter strains in broiler flocks and the potential involvement of poultry transport crates in transmitting these bacteria. This study also suggests that ineffective cleaning and disinfection procedures can increase Campylobacter contamination and facilitate the spread of bacteria in poultry establishments

Detection virulence factors and antimicrobial resistance patterns in Campylobacter spp. humans isolates and from slaughterhouses in southern brazil

Campilobacteriose é uma zoonose de distribuição mundial, com repercussões importantes na saúde pública e um grande impacto socioeconômico. O objetivo deste estudo foi investigar a ocorrência, padrões de resistência antimicrobiana e sua relação fenotípica e genotípica, bem como a caracterização de marcadores de virulência em isolados de Campylobacter spp. obtidos a partir de fontes de origem aviária de diferentes pontos na linha do abate de matadouros-frigoríficos do Estado do Rio Grande do Sul. Um total de 141 amostras, incluindo fezes (n=8), água de chiller (n=18), carcaças de frango durante o processo do abate (n=26) e carne de frango pronta para o consumo (n=89) foram avaliadas. Todos os isolados foram confirmados pela técnica m-PCR baseados na detecção da região 16S rRNA e os genes ceuE e mapA. Determinou-se a presença de Campylobacter jejuni em 140 amostras (99.2%), enquanto que Campylobacter coli foi identificado na amostra restante (0,7%). Cento e quarenta e uma cepas de Campylobacter spp. foram submetidas à analise de PCR para a detecção de marcadores de resistência e as 140 de Campylobacter jejuni para avaliar genes de patogenicidade. Em referência a Campylobacter jejuni, os resultaram indicaram que o gene flaA estava presente em 78.5% e o marcador cadF foi detectado em 77.8% dos isolados. Do total das amostras 85% (119/140) foram detectados para o gene cdtA, 80% (112/140) para o gene cdtB e 92.1% (129/140) para o gene cdtC. O operon (cdtABC) associado com a expressão total da toxina citoletal estava presente em 74.2% (104/140) das amostras. O marcador genético associado à invasão (iam) não foi encontrado em nenhum dos isolados de Campylobacter jejuni, e a ocorrência dos genes virB11 e wlaN foi de 22.1% e 10.7%, respectivamente. Na pesquisa de resistência a antimicrobianos, uma alta porcentagem (65%) (91/141) dos isolados de Campylobacter spp. são resistentes a β-lactâmicos. Cinquenta cepas (35.5%) são resistentes a tetraciclinas e 26 (18.5%) tem a presença da bomba de efluxo. Neste contexto, 36 de 141 cepas de Campylobacter (25.6%) são resistentes a dois diferentes marcadores de resistência (blaOXA-61 e tetO). Realizou-se também outro estudo, para detectar a mutação no gene gyrA da região determinante de resistência a quinolonas (QRDR). Um total de 50 amostras de Campylobacter jejuni foram submetidas a testes de sensibilidade mediante ensaios genotípicos e fenotípicos. A Concentração Inibitória Mínima (CIM) foi determinada utilizando a técnica de microdiluição em caldo. Os resultados obtidos mostraram uma porcentagem de 98% sensíveis a eritromicina. Em contraste, 94% de Campylobacter jejuni isolados foram resistentes à ciprofloxacina (47/50) e quarenta e cinco cepas (90%) resistentes ao ácido nalidíxico. Em referência aos isolados resistentes à ciprofloxacina, 100% das estirpes apresentavam relação entre o fenótipo de resistência e uma mutação no aminoácido 86 do gene gyrA, sendo detectada pelo ensaio de PCR-RFLP e posteriormente confirmada por sequenciamento. Os resultados deste estudo mostram uma grande diversidade entre os isolados analisados. O esforço para reduzir as infecções por Campylobacter spp. em humanos está diretamente ligado a uma melhor compreensão dos aspectos biológicos deste microrganismo e, particularmente, dos seus mecanismos de virulência e resistência, que contribuem na patogênese da doença.Campylobacteriosis is a zoonosis of worldwide distribution, with important implications for public health and a significant socioeconomic impact. The aim of this study was to investigate the occurrence, antimicrobial resistance patterns and phenotypic and genotypic relationship of Campylobacter species. A total of 141 samples, including feces (n = 8), chiller tank processing water (n = 18), carcasses during the slaughter process (n = 26) and poultry meat (n = 89) were evaluated. All the isolates were confirmed by m-PCR based detection of 16SrRNA, ceuE and mapA genes. The most ubiquitous of the thermotolerant Campylobacter spp. was C. jejuni. It was found in 140 of the contaminated samples (99.2%), whereas C. coli was identified in the remaining sample (0.7%). One hundred and forty-one strains of Campylobacter spp. analysis were subjected to PCR for the detection of resistance markers and 140 Campylobacter jejuni strains to evaluate pathogenic genes. In reference to Campylobacter jejuni, the obtained results showed that, the occurrence of flaA gene was 78.5% and cadF gene 77.8% in the isolates. The cytotoxin encoding cluster cdtABC was detected in 74.2% isolates. The frequency rates found for cdtA, cdtB and cdtC was 85%, 80%, and 92.1% respectively. The invasion-associated marker (iam) gene was not found in any of the Campylobacter isolates, and the occurrence of plasmidial virB11 and wlaN genes were 22.1%, and 10.7%, respectively. The results obtained point to the high percentage (65%) of Campylobacter isolates resistant to β-lactam. Fifty strains (35.5%) were resistant to tetracycline and 26 (18.5%) to the efflux pump. Moreover, 36 out of the 141 Campylobacter strains (25.6%) were found to be resistant to at least two different antimicrobial resistance markers (blaOXA-61 and tetO). Also, a total of 50 samples were screened for presence of antimicrobial sensibility for genotypic and phenotypic methods. Determination of Minimal Inhibitory Concentration (MIC) is tested using the standard microdilution method. The MICs results to 3 antimicrobial agents analyzed, showed that 98% of isolates were sensitive to erythromycin. In contrast, most isolates were resistant to ciprofloxacin (94%) and nalidixic acid (90%). Regarding ciprofloxacin-resistant isolates, 100% of the phenotype resistance strains had a mutation in the gyrA gene that was detected by the PCR-RFLP assay and sequencing. The results of this study show a great diversity among the isolates analyzed. The effort to reduce infections by Campylobacter spp. in humans is directly linked to a better understanding of the biological aspects of this microorganism and, particularly, its virulence and resistance mechanisms that contribute to the pathogenesis of the disease

Participação de genes associados ao processo de respiração anaeróbica na infecção de aves por Salmonella Typhimurium

Patogenos intestinais são expostos a várias condições de estresse durante o ciclo de infecção. Anaerobiose é uma condição hostil oferecida pelo hospedeiro no intestino e lúmen intestinal, onde a sobrevivência, multiplicação e entrada nas células epiteliais é prioridade para a invasão do agente patogênico. A fumarato reductase (frdABCD), dimetil sulfóxido (DMSO)- Nóxido de trimetilamina (TMAO) reductase (dmsABC), e nitrato reductase (narGHIJ), são genes de Salmonella Typhimurium que codificam enzimas envolvidas na respiração anaeróbica de fumarato, DMSO ou TMAO, e nitrato, respectivamente. Eles são regulados em resposta à disponibilidade de nitrato, oxigênio e a taxa de crescimento celular. A vitamina B12 (cobalamina) é sintetizada por Salmonella Typhimurium em condições anaeróbicas, sendo usado como cofator em quatro reações conhecidas. A deleção dos genes cobS e cbiA inibem a produção de cobalamina. No presente estudo, foi avaliada a infecção de aves por mutantes de STM, com o sistema respiratório comprometido por alterações nos genes narG, napA, cobS, cbiA, frdA, dmsA e torC. Aves de um dia de idade, foram inoculadas com 0.1 mL de cultura de Salmonella Typhimurium contendo 108 UFC/mL, diluída a 10-2 e 10-3. Sinais clínicos e mortalidade foram avaliados durante 21 dias. Em geral, os sintomas das aves infectadas com as cepas mutantes foram semelhantes aos apresentados pelas aves do grupo controle. Com exceção de STM cbiA, os demais mutantes reduziram a capacidade de causar mortalidade em comparação com a cepa original. A mortalidade do grupo de aves infectadas com STM ΔnarG, STM ΔfrdA, STM ΔdmsA e STM ΔcobSΔcbiA, apresentaram diminuição significativa da mortalidade em comparação ao grupo controle (p<0.05)Intestinal pathogens are exposed to various stress conditions during their infectious cycle. Anaerobiosis, one of such hostile condition, is offered by the host within gut and intestinal lumen, where survival, multiplication and entry into intestinal epithelial cells are priority for the invasion of the pathogen. The fumarate reductase (frdABCD), dimethyl sulfoxide (DMSO)- trimethylamine N-oxide (TMAO) reductase (dmsABC), and nitrate reductase (narGHIJ) operons in Salmonella Typhimurium (STM) encode enzymes involved in anaerobic respiration to the electron acceptors fumarate, DMSO, TMAO, and nitrate, respectively. They are regulated in response to nitrate and oxygen availability and changes in cell growth rate. Vitamin B12 (cobalamin) is synthesized by Salmonella Typhimurium only under anaerobic growth conditions used as a cofactor in four known reactions. The deletion of cobS and cbiA genes prevent any form of cobalamin production. In the present study we evaluate the infection of birds by mutants of STM, with the anaerobic respiratory system committed by mutations in the genes: narG, napA, cobS, cbiA, frdA, dmsA, and torC. Virulence was assessed by oral inoculation of groups of one-day-old broilers with 0.1 mL of culture contained 108 CFU/mL or diluted at 10-3 and 10-2 of strains mutants of Salmonella Typhimurium. Clinical signs and mortality were recorded over a period of 21 days. In general, the symptoms of chickens infected with the mutant strains were similar to those presenting by control birds. Except for STM ΔcbiA, all showed reduced capacity to cause mortality in comparison with the original strain. The mortality of group of chickens infected with STM ΔnarG, STM ΔfrdA, STM ΔdmsA and STM ΔcobSΔcbiA showed significant decrease in mortality compared to control group (p<0.05)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Boletín del Servicio Meteorológico Nacional: Epoca 2ª Año XVII Número 190 - 1968 Julio 08

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A defective mutant of Salmonella enterica Serovar Gallinarum in cobalamin biosynthesis is avirulent in chickens

Salmonella enterica serovar Gallinarum (SG) é o agente do tifo aviário, doença severa que provoca mortalidade em até 80% do plantel de aves. SG encontra-se entre os poucos sorotipos de Salmonella que são agentes etiológicos de enfermidade específica, à semelhança de Salmonella Typhi em seres humanos podendo, portanto, servir de modelo experimental para outras salmoneloses hospedeiro especíifcas. Além dos mecanismos de virulência, a bactéria utiliza mecanismos de sobrevivência para permanecer no hospedeiro. A ativação desses mecanismos pode ou não estar associada à ativação dos mecanismos de virulência. Entre os mecanismos fisiológicos, está a produção de vitamina B12 que Salmonella spp. realiza em ambientes anaeróbicos, como quando encontra-se intracelularmente no organismo hospedeiro. Neste estudo, analisou-se a infecção de aves por cepas de SG, que tiveram genes alterados que participam da biossíntese de vitamina B12. Foram produzidos mutantes de SG contendo os genes cbiA e cobS alterados e um terceiro, contendo ambos os genes alterados. A sobrevivência e a ação patogênica de SG não foi modificada pela alteração simples de um dos genes, mas tornou a cepa de SG completamente atenuada quando os dois foram modificados. A mortalidade provocada pela cepa selvagem de SG foi de 64,52%, enquanto que não observou-se mortalidade alguma no grupo de aves infectadas com SGNal rcobscbiA. Estudos futuros deverão ser realizados para elucidar este processo fisiológico bacteriano e para avaliar a utilização desta cepa de SG como cepa vacinal.Salmonella enterica serovar Gallinarum (SG) is a fowl typhoid agent in chickens and is a severe disease with worldwide economic impact as its mortality may reach up to 80%. It is one of a small group of serovars that typically produces typhoid-like infections in a narrow range of host species and which therefore represents a good model for human typhoid. The survival mechanisms are not considered to be virulent mechanisms but are essential for the life of the bacterium. Mutants of Salmonella Gallinarum containing defective genes, related to cobalamin biosynthesis and which Salmonella spp. has to be produced to survive when it is in an anaerobic environment, were produced in this study. Salmonella Gallinarum is an intracellular parasite. Therefore, this study could provide information about whether vitamin B12 biosynthesis might be essential to its survival in the host. The results showed that the singular deletion in cbiA or cobS genes did not interfere in the life of Salmonella Gallinarum in the host, perhaps because single deletion is not enough to impede vitamin B12 biosynthesis. It was noticed that diluted SG mutants with single deletion produced higher mortality than the wild strain of SG. When double mutation was carried out, the Salmonella Gallinarum mutant was unable to provoke mortality in susceptible chickens. This work showed that B12 biosynthesis is a very important step in the metabolism of Salmonella Gallinarum during the infection of the chickens. Further research on bacterium physiology should be carried out to elucidate the events described in this research and to assess the mutant as a vaccine strain.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Campylobacter jejuni e C. coli em carcaças de frangos após a refrigeração por imersão no sul do Brasil

Campylobacter jejuni and C. coli have been associated with gastrointestinal disorders in human beings, due mainly to the consumption of chicken meat. Despite control measures for reducing contamination by these bacteria, the detection of Campylobacter in carcasses after chilling remains high. A total of 105 carcasses were assessed by the horizontal detection method in five federally inspected slaughterhouses in southern Brazil in 2012 and in the first three months of 2013. Campylobacter was isolated in 37.1% of the carcasses, of which 97.5% contained C. jejuni and 2.5% were infected by C. coli. The rate of positive carcasses across the slaughterhouses ranged from 0 to 71.4%. Determining the occurrence of Campylobacter among flocks is crucial for estimating the microbial load at specific points along the slaughtering process and for minimizing the risk of contamination of end products by Campylobacter.Campylobacter jejuni e C. coli têm sido associados a problemas gastroentéricos em seres humanos principalmente devido ao consumo de carne de frango. Embora medidas de controle sejam realizadas para reduzir a contaminação por estas bactérias, a identificação de Campylobacter em carcaças após a refrigeração por imersão é alto. Foram analisadas 105 carcaças pelo método de detecção horizontal em cinco abatedouros sob Inspeção Federal no sul do Brasil em 2012 e nos três primeiros meses de 2013. Campylobacter foi isolada em 37,1% das carcaças analisadas, as quais 97,5% foram identificados como C. jejuni e 2,5% como C. coli. A ocorrência de carcaças positivas entre matadouros variou de zero a 71,4%. O conhecimento sobre a ocorrência de Campylobacter entre os lotes é fundamental para estimar a extensão da carga microbiana em pontos específicos do abate e consequentemente minimizar o risco de contaminação por Campylobacter em produtos finais de frangos