Determination of multidrug resistance mechanisms in Clostridium perfringens type A isolates using RNA sequencing and 2D-electrophoresis

In this study, we screened differentially expressed genes in a multidrug-resistant isolate strain of Clostridium perfringens by RNA sequencing. We also separated and identified differentially expressed proteins (DEPs) in the isolate strain by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The RNA sequencing results showed that, compared with the control strain, 1128 genes were differentially expressed in the isolate strain, and these included 227 up-regulated genes and 901 down-regulated genes. Bioinformatics analysis identified the following genes and gene categories that are potentially involved in multidrug resistance (MDR) in the isolate strain: drug transport, drug response, hydrolase activity, transmembrane transporter, transferase activity, amidase transmembrane transporter, efflux transmembrane transporter, bacterial chemotaxis, ABC transporter, and others. The results of the 2-DE showed that 70 proteins were differentially expressed in the isolate strain, 45 of which were up-regulated and 25 down-regulated. Twenty-seven DEPs were identified by MS and these included the following protein categories: ribosome, antimicrobial peptide resistance, and ABC transporter, all of which may be involved in MDR in the isolate strain of C. perfringens. The results provide reference data for further investigations on the drug resistant molecular mechanisms of C. perfringens.

Antimicrobial activities of six essential oils commonly used as condiments in Brazil against Clostridium perfringens

Abstract Despite recent advances in food production technology, food-borne diseases (FBD) remain a challenging public health concern. In several countries, including Brazil, Clostridium perfringens is among the five main causative agents of food-borne diseases. The present study determines antimicrobial activities of essential oils of six condiments commonly used in Brazil, viz., Ocimum basilicum L. (basil), Rosmarinus officinalis L. (rosemary), Origanum majorana L. (marjoram), Mentha × piperita L. var. Piperita (peppermint), Thymus vulgaris L. (thyme) and Pimpinella anisum L. (anise) against C. perfringens strain A. Chemical compositions of the oils were determined by GC–MS (gas chromatography–mass spectrometry). The identities of the isolated compounds were established from the respective Kováts indices, and a comparison of mass spectral data was made with those reported earlier. The antibacterial activity was assessed from minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using the microdilution method. Minimum inhibitory concentration values were 1.25 mg mL-1 for thyme, 5.0 mg mL-1 for basil and marjoram, and 10 mg mL-1 for rosemary, peppermint and anise. All oils showed bactericidal activity at their minimum inhibitory concentration, except anise oil, which was only bacteriostatic. The use of essential oils from these common spices might serve as an alternative to the use of chemical preservatives in the control and inactivation of pathogens in commercially produced food systems.

Caracterización molecular y resistencia antimicrobiana de aislamientos de Clostridium perfringens de diferentes orígenes en Costa Rica

Molecular characterization and antimicrobial resistance of Clostridium perfringens isolates of different origins from Costa Rica. Clostridium perfringens, a Gram positive, spore-forming anaerobe, is widely distributed in nature. Based upon their production of four major toxins α, β, ε and ι, C. perfringens is classified into five toxinotypes (A-E). Some strains produce an enterotoxin (CPE), encoded by the cpe gene, which causes diarrhea in humans and some animals. C. perfringens strains that had been previously isolated and been kept at -80°C were analyzed for the presence of toxin genes and for antimicrobial resistance: 20 from soils,20 from animal, 20 from human origin and 21 from food non related to outbreaks. According to PCR results, all strains were classified as C. perfringens type A, since only α toxin gene was detected, while cpe was detected in two strains (2.5%) isolated from food, as it has been described in other world regions. Antibiotic resistance to at least one antibiotic was detected in 44% of the strains, 41% was resistant to clindamycin, 25% to chloramphenicol, 22% to penicillin and 20% to metronidazole. Soils strains showed the highest resistance percentages to almost all antibiotics. Multiresistance (to three or more antibiotic groups) was detected in the strains from soil (40%), human origin (30%), food (14%) and animal origin (5%). The high resistance rates found may be explained by the widespread use of antimicrobials as growth promoters in plants and animals; also these resistant strains may act as reservoir of resistance genes that may be transferred between bacteria in different environments. Rev. Biol. Trop. 59 (4): 1479-1485. Epub 2011 December 01.

Clostridium perfringens es un bacilo Gram positivo, esporulado, anaerobio, ampliamente distribuido en la naturaleza, que produce cuatro toxinas principales α, β, ε y ι, las cuales permiten su clasificación en cinco toxinotipos (A-E). Algunas cepas producen una enterotoxina (CPE), codificada por el gen cpe, que causa diarrea en seres humanos y en algunos animales. La presencia de los genes de estas toxinas y la sensibilidad a los antibióticos se determinó en 81 cepas de C. perfringens previamente aisladas y que habían sido mantenidas a -80°C; 20 de suelos, 20 de origen animal, 20 de origen humano y 21 de alimentos cocidos no relacionados con brotes alimentarios. De acuerdo con los resultados de PCR, todas las cepas fueron clasificadas como C. perfringens tipo A, debido a que solo se les detectó el gen de la toxina α, mientras que el gen de la enterotoxina (cpe) se detectó en dos cepas (2.5%) aisladas de alimentos, tal como ha sido descrito en otras regiones del mundo. El 44% de las cepas fue resistente a algún antibiótico; clindamicina (41%), cloranfenicol (25%), penicilina (22%) y metronidazol (20%). En general, las cepas provenientes de suelos presentaron los mayores porcentajes de resistencia a casi todos los antibióticos. El 40% de las cepas de suelo presentó multiresistencia (a tres o más grupos de antibióticos), el 30% de las de origen humano, el 14% de las de alimentos y el 5% de las de origen animal. Las altas tasas de resistencia encontradas podrían deberse al amplio uso de antibióticos como promotores de crecimiento de plantas y animales y esas cepas resistentes podrían actuar como reservorio de genes de resistencia que pueden transferirse entre bacterias de diversos ambientes.

Antimicrobial resistance among Clostridium perfringens isolated from various sources in Thailand.

Antimicrobial resistance among Clostnridium perfringens isolated from feces of humans and pigs, food and other environmental sources was examined by testing of 201 PCR-confirmed strains for resistance to 7 antimicrobial agents. The minimal inhibitory concentrations (MICs) were determined by the agar dilution method. Overall, C. perfringens showed the highest resistance to tetracycline (56.2%), followed by imipenem (24.9%), metronidazole (9.5%), penicillin G (9%), vancomycin (4.5%), chloramphenicol (3%) and ceftriaxone (1%). The majority of the isolated strains from pig feces (77.8%), environment (72.7%), human feces (44.9%) and food (28%) showed resistance to tetracycline. Strains isolated from human feces only showed low resistance to ceftriaxone (2.5%) and vancomycin (10.1%). Penicillin G had high activity, with overall MIC50 and MIC90 of 0.06 and 1.0 microg/ml, respectively, and low rate of resistance (10-12% for strains isolated from humans, animals and food). Among 62.7% of antimicrobial resistant strains, 39.3% were resistant to a single drug and 23.4% were multiple-drug resistant (MDR). Of overall 47 MDR strains, 63.8% were derived from human feces and were resistant to two to six drugs.

Nitroimidazoles, Part XXIII--activity of satranidazole series against anaerobic infections.

A large number of nitroimidazoles have been examined for in vitro activity against three anaerobes - Bacteroides fragilis (Bf), a strain of Bf resistant to metronidazole (16a) and Clostridium perfringens and many found to be active. Among these may be mentioned 1-methyl-5-nitroimidazoles carrying N - bound hetetocycles at position 2, such as satranidazole 1a, 1b, 1c, 1k, 1n and 1v which are at least twice as active as metronidazole (16a), ornidazole (16b) and tinidazole (16c). Even more active are 5-nitroimidazolyl benzimidazole 5d, -thiazolidinone 6b and thiadiazolidine dioxide 8a. Many other types of compounds derived from 1-methyl-2-amino-5-nitroimidazole are feebly active. Among 5-nitroimidazoles with a carbon substituent at position 2, 16a, 16b and 16c are equiactive while dimetridazole 14f is more active than 16a against Bf. Some 2-vinyl derivatives are very potent, with 18f and 18i being outstanding. Activity better than that of metronidazole is seen for nitroimidazooxazepines, e.g. 29d. 5-Nitroimidazoles are more active against anaerobes than 4-nitro isomers. Antianaerobic and antiamoebic activities generally run parallel in these classes of compounds. The study has led to the elaboration of the antianaerobic profile of satranidazole 1a.

Studies on the alpha toxin of clostridium perfringens type A in pigs

Se estudió si la toxina alfa desmpeña un papel en la enteritis del cerdo producida por C. perfringens tipo A. De las cepas aisladas de C, perfringens tipo A, 38 de 42 produjeron niveles detectables de toxina alfa con títulos de a a 256 unidades hemolíticas. La toxina alfa fue purificada a partir de una de esas cepas por medio de un procedimiento en 3 pasos, encontrándose libre de contaminantes. La toxina purificada se utilizó para la producción de una antitoxina, preparada en conejos y utilizada en el estudio de sus efectos en asas intestinales del cerdo en donde se observaron ligeros cambios histológicos que incluyen congestión de la mucosa y una hipercelularidad localizada en la lámina propia; los efectos de la toxina alfa fueron neutralizados por su antitoxina. En poblaciones sanas 45 de 106 sueros de cerdos presentaron títulos de anticuerpos antotoxina alfa de 1:2 a 1:32. Los resultados indicaron que cepas de C. perfringens tipo A productoras de toxina alfa se encuentran ampliamente diseminadas en las poblaciones porcinas y sugieron que la toxina alfa desempeña un papel dentro de la patogénesis de la enteritis producida por C. perfringens tipo A

Sporulation and enterotoxin production by lostridium perfringenstype a strains isolated from pig diarrhea

42 cepas de C. perfringens tipo A aislada de casos de diarrea en cerdos se probaron en su capacidad de esporular y producir enterotoxina en 3 diferentes medios promotores de esporulación. 11 de las 42 (26.1 por ciento) cepas de C. perfringens tipo a produjeron enterotoxina. 13 cepas (30.9 por ciento) produjeron esporas con niveles del 10 por ciento o mas del total de celulas bacterianas. 24 cepas (57.1 por ciento) fueron capaces de esporular. Los titulos de enterotoxina producidos por las cepas variaron entre 1:2 a 1:64. La enterotoxina producida por una de las cepas fue purificada y se produjo un antisuero en conejos. Esporas de C. perfringens tipo A se detectaron en heces en 20 de 23 casos de enteritis con niveles de hastas 5 x 10**6 células por gramo de materia fecal. Cuentas mas bajas de esporas de hasta 2 x 10**4 células por gramo de materia fecal se encontraron en 5 de 10 muestras procedentes de cerdos sanos. La enterotoxina se detecto en cultivo de células Vero en 5 de las 23 muestras procedentes de cerdos con diarrea; las 10 muestras de cerdos sanos se encontraron negativas a la presencia de enterotoxina. Estos estudios indican que cepas de C. perfringes tipo A son capaces de esporular y producir enterotoxina in vitro e in vivo y que el cuadro diarréico observado puede estar relacionado con la esporulación de las bacterias in vivo

Teste alternativo de sensibilidade de bactérias anaeróbias estritas a agentes antimicrobianos / Alternative susceptibility test of anaerobic bacteria to antimicrobial agents

Um total de 25 amostras de bactérias anaeróbias, Bacteroides fragilis, Clostridium perfringens e Clostridium difficile foram avaliadas quanto a sensibilidade a vários antimicrobi anos pelo método de eluiçäo do disco em caldo BHI pré-reduzido esterilizado em anaerobiose (PRAS) e os resultados foram comparados com aqueles obtidos por uma metodologia de BHI näo-PRAS. Uma grande correlaçäo entre os dois métodos justifica a indicaçäo desta alternativa como procedimento satisfatório em rotina bacteriológica