ABSTRACT
Abstract Clostridioides difficile is a spore-forming anaerobe microorganism associated to nosocomial diarrhea. Its virulence is mainly associated with TcdA and TcdB toxins, encoded by their respective tcdA and tcdB genes. These genes are part of the pathogenicity locus (PaLoc). Our aim was to characterize relevant C. difficile toxinotypes circulating in the hospital setting. The tcdA and tcdB genes were amplified and digested with different restriction enzymes: EcoRI for tcdA; HincII and AccI for tcdB. In addition, the presence of the cdtB (binary toxin) gene, TcdA and TcdB toxins by dot blot and the cytotoxic effect of culture supernatants on Vero cells, were evaluated. Altogether, these studies revealed three different circulating toxinotypes according to Rupnik's classification: 0, I and VIII, being the latter the most prevalent one. Even though more studies are certainly necessary (e.g. sequencing analysis), it is worth noting that the occurrence of toxinotype I could be related to the introduction of bacteria from different geographical origins. The multivariate analysis conducted on the laboratory values of individuals infected with the most prevalent toxinotype (VIII) showed that the isolates associated with fatal outcomes (GCD13, GCD14 and GCD22) are located in regions of the biplots related to altered laboratory values at admission. In other patients, although laboratory values at admission were not correlated, levels of urea, creatinine and white blood cells were positively correlated after the infection was diagnosed. Our study reveals the circulation of different toxinotypes of C. difficile strains in this public hospital. The variety of toxinotypes can arise from pre-existing microorganisms as well as through the introduction of bacteria from other geographical regions. The existence of microorganisms with different pathogenic potential is relevant for the control, follow-up, and treatment of the infections.
Resumen Clostridioides difficile es un anaerobio esporulado que se asocia con episodios de diarreas hospitalarias. Su virulencia se encuentra vinculada, principalmente, a las toxinas TcdA y TcdB, codificadas por sus respectivos genes, tcdA y tcdB, que son parte de un locus de patogenicidad (PaLoc). Nuestro objetivo fue caracterizar los toxinotipos de C. difficile circulantes en un hospital público. Los genes tcdA y tcdB fueron amplificados y digeridos con diferentes enzimas de restricción: EcoRI para tcdA; HincII y AccI para tcdB. Además, se evaluó la presencia de cdtB (gen de la toxina binaria B) y de las toxinas A y B (por dot blot), así como el efecto citotóxico de sobrenadantes de cultivo sobre células Vero. En conjunto, estos estudios revelaron tres toxinotipos circulantes según la clasificación de Rupnik: 0, I y VIII; el más prevalente fue el último. Aunque son necesarios más estudios (ej., secuenciación), es interesante notar que la presencia del toxinotipo I podría estar relacionada con la introducción de bacterias de diferente origen geográfico. En los pacientes infectados con el toxinotipo VIII, el análisis multivariante de los resultados de laboratorio mostró que los aislamientos asociados a decesos (GCD13, GCD14 y GCD22) estaban situados en regiones de los biplots relacionados con valores de laboratorio alterados al momento de la internación. En los otros pacientes, aunque no se observó correlación entre los valores de laboratorio al momento de la internación y la evolución clínica, los niveles de urea, creatinina y recuento de glóbulos blancos estuvieron correlacionados positivamente entre sí una vez diagnosticada la infección. Nuestro estudio revela la circulación de diferentes toxinotipos de C. difficile en un mismo hospital público. La variedad de toxinotipos puede originarse a partir de microorganismos preexistentes en la región, así como también por la introducción de bacterias provenientes de otras regiones geográficas. La existencia de microorganismos con diferente potencial patogénico es relevante para el control, el seguimiento y el tratamiento de las infecciones.
ABSTRACT
Clostridium perfringens is well known causative agent of necrotic enteritis in poultry and is mainly caused by Type A toxin. NetB toxin is found to be one of the newly emerging virulent toxin gene which is also responsible for necrotic enteritis. The present study was carried out to characterize and to type the different toxins associated with C. perfringens in NE cases of poultry. For the present study total 125 samples were collected from poultry birds, out of which 50 samples were of intestinal content from diseased birds, 50 cloacal swabs and 25 intestinal content from healthy birds. These samples were further processed for isolation, identification, and toxinotyping of Clostridium perfringens isolates. Onisolation of C. perfringens on blood agar total 43 isolates were found positive showing a pattern of double hemolysis on blood agar. The positive isolates of C. perfringens were further confirmed by using 16S rRNA species specific PCR. After confirmation isolates were processed for toxinotyping mainly targeting cpa, cpb and cpb2 toxins by using multiplex PCR. On toxinotyping it was found that NE in poultry birds were mainly caused by C. perfringens type A. On virulent gene detection of netB toxin, total 4 isolates were found positive for netB toxin. This study pointed out that C. perfringens type A is responsible for development of NE in poultry along with net B toxin which is a new key virulent factor. Further studies of netB toxoid and C. perfringens type A for vaccine production could minimize the clostridial problems in broiler farms
ABSTRACT
Clostridium perfringens (Cp) é uma bactéria aneróbica gram positiva que, além de provocar gangrena gasosa e enterotoxemia em humanos e animais, constitui-se na principal causa de enterite necrótica em aves de criações intensificadas. A identificação dos isolados foi realizada pela reação de lecitinase em ágar TSC-gema de ovo, colônias com dupla hemólise em ágar sangue desfibrinado de eqüino, coloração de Gram e provas bioquímicas. Das amostras analisadas, 171Cp foram isolados em jejuno e íleo de frangos de corte provenientes de um frigorífico da região de Pará de Minas-MG. Cp foi isolado em 62 (49,6 por cento) de 125 amostras de conteúdo lumenal de jejunos e em 109 (87,2 por cento) de igual número de íleos dos frangos de corte. Utilizando-se a técnica da PCR múltipla para genotipicacão das estirpes de Cp, de acordo com os genes para as toxinas principais e letais (cpa, cpb, etx e iA), da toxina cpb2 (cpb2) e enterotoxina (cpe), as estirpes de Cp isoladas foram classificadas em cinco tipos toxigênicos (A-E). Das 62 estirpes de Cp isoladas do jejuno, foram obtidos 42/62 (67,7 por cento) tipo A, 1/62 (1,6 por cento) tipo A com produto de amplificação para o gene da toxina beta2, 0/62 (0 por cento) tipo B, 17/62 (27,4 por cento) tipo C, 1/62 (1,6 por cento) tipo D. Das 109 amostras de Cp isolados do íleo das aves foram obtidos 62/109 (56,9 por cento) tipo A, 3/109 (2,7 por cento) tipo A com produto de amplificação para o gene da toxina beta2, 1/62 (0,9 por cento) tipo B, 38/109 (34,9 por cento) tipo C, 1/109 (0,9 por cento) tipo D. Cp A (60,8 por cento) e Cp C (32,2 por cento) foram os tipos toxigênicos predominantes em conteúdo intestinal de frango de corte. Cinco (2,9 por cento) das 171 amostras de Cp isolados não foram tipificadas. Não foram detectados os genes codificadores das toxinas iota (iA) e enterotoxina (cpe) em nenhuma das 171 estirpes de Cp caracterizados.
Clostridium perfringens (Cp) is an anaerobic gram-positive bacterium which causes gaseous gangrene and enterotoxaemias in humans and domestic animals, besides being the primary cause of necrotic enteritis in poultry. Cp isolates were preliminary identified according to the lecithinase test on agar TSC-egg yolk, colony with double haemolysis in desfibrinated horse blood agar, Gram staining and biochemical tests. Cp isolates (171) were obtained from the intestinal content of broiler chickens sampled in a slaughterhouse in Pará de Minas city, MG, Brazil. Cp was isolated in 62/125 (49.6 percent) strains from jejunum content and in 109/125 (87.2 percent) of ileum. Cp strains were classified into five toxigenic types (A-E), using multiplex PCR assay for genotyping of the principal and lethal toxins in the detection of genes coding for toxins alfa, beta, epsilon e iota, nomely genes cpa, cpb, etx e iA genes, beta2 toxin (cpb2) and enterotoxin (cpe). From a total of 62 Cp jejunum isolates obtained 42/62 (67.7 percent) were type A, 1/62 (1.6 percent) type A with the amplification of products for beta2 toxin gene (A/B2), 0/62 (0 percent) type B, 17/62 (27.4 percent) type C and 1/62 (1.6 percent) type D. A total of 109 ileum Cp isolates were obtained being 62/109 (56.9 percent) type A, 3/109 (2.7 percent) type A/B2 toxin gene, 1/62 (0.9 percent) type B, 38/109 (34.9 percent) type C, 1/109 (0.9 percent) type D. Cp A (60.8 percent) and Cp C (32.2 percent) toxigenic types were the most prevalent types in the analyzed intestinal contents of broiler chickens Cp A 104/171 (60.8 percent) and 55/171 (32.2 percent) toxigenic types which were the most prevalent types analyzed into two partes of the intestinal content of broiler chickens. Five (2.9 percent) out of 171 Cp isolates were not typified. The iota toxin (iA) and enterotoxin gene (cpe) codifying genes were not identified.