Unión de los antibióticos tilosina, tilmicosina y oxitetraciclina a proteínas presentes en abejas, larvas y productos de la colmena de Apis mellifera L / Binding of tylosin, tilmicosin and oxytetracycline to proteins from honeybees, larvae and beehive products

Las abejas melíferas son afectadas por gran cantidad de enfermedades infecciosas principalmente producidas por bacterias, hongos, virus y parásitos eucariotas. Dentro de las ocasionadas por procariotas, la loque americana es una enfermedad extremadamente grave que afecta a larvas y pupas de abejas; su agente causal es la bacteria esporulada Paenibacillus larvae. La administración de antibióticos es la principal alternativa para el control de esta enfermedad en colmenares con altos niveles de infección. El objetivo del presente trabajo fue determinar, mediante un método biológico, la unión de los antibióticos tilosina, tilmicosina y oxitetraciclina a las proteínas presentes en abejas adultas, larvas menores de 72 horas, larvas mayores de 72 horas, jalea de obreras, miel y polen, con la finalidad de diseñar un modelo de ruta cinética de los antibióticos. Los límites de sensibilidad de la técnica de valoración de estos antibióticos fueron 0,05 μg/ml para tilosina y tilmicosina, y 0,01 μg/ml para oxitetraciclina. Los coeficientes de correlación fueron superiores a 0,90 y los coeficientes de variación intra e inter-ensayo inferiores al 5%. Tanto tilosina como oxitetraciclina presentaron un porcentaje de unión a proteínas de un 15% en promedio en tejidos y subproductos de la colmena, lo cual resultó inferior a lo observado con tilmicosina (29% en promedio). En conclusión, por sus características químicas, su actividad antimicrobiana y su baja tasa de unión a las abejas, larvas y subproductos de la colmena, la tilosina presenta propiedades farmacocinéticas que podrían representar una ventaja terapéutica para el tratamiento de la loque americana en colmenas.

American Foulbrood (AFB) caused by the spore-forming bacterium Paenibacillus larvae is the most serious disease of bacterial origin affecting larvae and pupae of honeybees. Antibiotics are used in many countries for the control of AFB in high incidence areas, but their misuse may lead to antibiotic resistance of bacterial strains and honey contamination. The objective of the present work was to determine, through a biological method, the protein binding of tylosin, tilmicosin and oxytetracycline to worker jelly; honey; pollen; adult bees and larvae in order to propose their kinetic routes. The sensitivity limit of the technique used was 0.05 μg/ml for tylosin and tilmicosin and 0.01 μg/ml for oxytetracycline, respectively. The method had intra and inter-assay correlation coefficients over 0.90, respectively and a coefficient variation of intra-and inter-assay for all antibiotics and processed samples under 5%. Tylosin and oxytetracycline presented lower percentages of protein binding in tissues and hive products (average 15%) in relation to those observed for tilmicosin (29%). In conclusion, tylosin is useful for AFB control in honey bee colonies due to its chemical characteristics, antimicrobial activity and levels of protein binding in bees, larvae, and beehive products.

Enterotoxigenic gene profiles of Bacillus cereus and Bacillus megaterium isolates recovered from honey / Búsqueda de factores de virulencia en cepas de Bacillus cereus y de Bacillus megaterium aisladas de miel

One hundred and thirty two Bacillus cereus and 52 Bacillus megaterium isolates from honeys were evaluated for the presence of genes encoding enterotoxin HBL, enterotoxin-T, cytotoxin K and the NHE complex, respectively. The relationship between hemolytic and coagulase activity and its correlation with the presence of the four mentioned enterotoxins was determined by principal component analysis (PCA). PCA in B. cereus revealed a positive correlation among free coagulase, hemolysis and the presence of genes hblA, hblB, hblC, hblD (HBL complex) and bceT (enterotoxin-T), but no correlation with the clumping factor (bound coagulase) and the presence of sequences of the NHE complex. On the other hand, PCA in B. megaterium showed a high positive correlation between coagulase (bound and free) and the haemolytic activity but no correlation in relation to the presence of genes of the HBL complex, cytotoxin K, enterotoxin T and the NHE complex. To our knowledge, this is the first report of the detection of cytotoxin K and of the NHE complex genes in B. megaterium. The relationship between the coagulase activity and the presence of virulence factors has not been described before in the genus Bacillus, being this work the first report of this correlation. Interestingly, the presence of the cytK gene was almost independent of the presence of the rest of virulence factors herein analyzed both in B. cereus and B. megaterium populations. Our results suggest that honey could be a possible vehicle for foodborne illness due to the presence of toxigenic B. cereus and B. megaterium strains containing different virulence factors.

Se evaluaron 132 aislamientos de Bacillus cereus y 52 de Bacillus megaterium provenientes de mieles de distintos orígenes geográficos para investigar la presencia de secuencias de ADN relacionadas con genes de virulencia y su posible correlación con la actividad hemolítica y coagulasa. Con respecto a los genes de virulencia, se analizaron por PCR secuencias de ADN de los genes nhe (A, B y C), HBL (A, B, C, D), cytK y bceT. La relación entre las variables fue evaluada mediante un análisis de componentes principales, donde se encontró que los aislamientos de B. cereus mostraron una correlación positiva entre actividad de coagulasa (coagulasa libre) y presencia de los genes del complejo HBL y bceT, mientras que en B. megaterium se halló una alta correlación positiva entre actividad de coagulasa (libre y fija) y actividad hemolítica, pero no se observó correlación significativa entre la presencia de genes de virulencia y dichas actividades. Este estudio constituye el primer registro de la presencia de los genes cyt K y NHE en cepas de B. megaterium y el primer trabajo que analiza la relación entre la actividad de coagulasa y la presencia de genes de virulencia en B. cereus y B. megaterium. La presencia del gen cytK en ambas especies resultó totalmente independiente del resto de los factores de virulencia analizados. Nuestros hallazgos sugieren que la miel podría vehiculizar enfermedades transmisibles por alimentos debido a la presencia de cepas de B. cereus y B. megaterium potencialmente tóxicas.

Tetracycline and oxytetracycline resistance determinants detected in Bacillus cereus strains isolated from honey samples / Detección de determinantes de resistencia a tetraciclina y oxitetraciclina en cepas de Bacillus cereus aisladas de muestras de miel

The aim of this study was to investigate the presence of tetracycline and oxytetracycline resistance determinants in Bacillus cereus strains isolated from honey samples. Of a total of 77 isolates analyzed, 30 (39%) exhibited resistance to tetracyclines according to the results of a disk diffusion method. Resistant strains (n=30) were screened by PCR for the presence of the resistant determinants tetK, tetL, tetM, tetO, tetW, otrA and otrB and their MIC values for tetracycline, oxytetracycline and minocycline were assessed. According to the PCR results, 23 isolates (77%) presented at least one tetracycline or oxytetracycline resistance determinant. The tetK genotype was present in 10 isolates while the tetL, tetM, and otrA genotypes were present in 3, 2, and 5 isolates, respectively. In addition, 2 isolates of the tetK plus tetM genotype, 1 of the tetK plus tetL genotype, and 1 of the tetK plus otrA genotype were found. All isolates were tetW, tetO and otrB negatives. On the other hand, 7 isolates (23%) showed a tetracycline-resistant and/or minocyclineresistant phenotype (MIC) but did not carry any of the tet or otr determinants investigated in this study. This research has shown that B. cereus isolates from honey samples contain a variety of tetracycline and oxytetracycline resistance genes, including the tetK and tetL determinants which encode for efflux proteins, and tetM and otrA, which encode for ribosomal protection proteins. These findings indicate that strains isolated from honeys could represent a reservoir for tetracycline resistance genes. To our knowledge, this is the first report of tetracycline-resistant and oxytetracyclineresistant B. cereus strains carrying the tetK determinant, and also the first report of oxytetracycline-resistant and tetracycline- resistant Bacillus species carrying the otrA determinant.

El objetivo del presente estudio ha sido investigar la presencia de diversos determinantes de resistencia a tetraciclina y oxitetraciclina en las poblaciones de Bacillus cereus presentes en la miel. De un total de 77 aislamientos evaluados, 30 (39%) resultaron resistentes a tetraciclina y/o minociclina de acuerdo con los resultados de las pruebas de difusión en disco. Dentro del grupo que presentó un fenotipo resistente, se investigó la presencia de los determinantes tetK, tetL, tetM, tetO, tetW, otrA y otrB por PCR y se determinaron los valores de CIM para tetraciclina, oxitetraciclina y minociclina. De acuerdo con los resultados obtenidos por PCR, 23 aislamientos (77%) presentaron al menos un determinante de resistencia a tetraciclina o a oxitetraciclina; el genotipo tetK se encontró en 10 de esos aislamientos, mientras que los genotipos tetL, tetM y otrA se hallaron en 3, 2 y 5 aislamientos, respectivamente. Ningún aislamiento presentó los genotipos tetW, tetO ni otrB. Adicionalmente, se encontraron los genotipos tetK plus tetM (2 aislamientos); tetK plus tetL (1 aislamiento) y tetK plus otrA (1 aislamiento). Por otra parte, 7 cepas (23%) resultaron resistentes a tetraciclina, oxitetraciclina y/o minociclina por CIM, pero no presentaban ninguno de los determinantes tet u otr estudiados. Estos resultados indican la existencia de un alto porcentaje de cepas de B. cereus aisladas de miel con genes de resistencia a tetraciclina y oxitetraciclina, incluyendo los determinantes tetK, tetL, tetM y otrA. Este estudio constituye el primer registro de la presencia del determinante tetK de resistencia a tetraciclina en B. cereus, como así también la presencia del determinante otrA dentro del género Bacillus.

Inhibition of the growth of Ascosphaera apis by Bacillus and Paenibacillus strains isolated from honey / Inhibición del crecimiento de Ascosphaera apis mediante cepas de Bacillus y Paenibacillus aisladas de miel

The fungus Ascosphaera apis, the causative agent of chalkbrood disease in honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of 249 aerobic spore-forming bacterial antagonists isolated from honey samples was evaluated. Each isolate was screened against A. apis by a central disk test assay. Ten bacterial strains that showed the best antagonistic effect to A. apis were selected for further study and identified as Bacillus cereus (m363, mv86, mv81, mv75), Bacillus circulans (Fr231, m448b), Bacillus megaterium (m435), Bacillus pumilus (m354), Bacillus subtilis (m329), and Paenibacillus alvei (m321). For testing the efficiency of the selected strains, a paired culture test was used with 5 replicates of each combination bacterial antagonist / A. apis strain, and 5 replications for each control on 4 different culture media. The analysis of variance and posterior comparison of means according to LSD method showed that the best antagonists when using YGPSA medium were B. subtilis (m329) and B. megaterium (m435), and in the case of MYPGP medium the most efficient were B. circulans strains Fr 231 and m448b.

La cría yesificada es una micosis invasiva ocasionada por el hongo heterotálico Ascosphaera apis que afecta exclusivamente a las larvas de las abejas. La enfermedad tiene difusión mundial y en la Argentina se halla diseminada en todas las áreas donde se realiza apicultura. Se estudió la potencialidad de 249 cepas de bacterias esporuladas aeróbicas aisladas de miel como agentes biocontroladores del hongo mediante un ensayo en disco central en condiciones de laboratorio. Se seleccionaron como mejores antagonistas 10 cepas bacterianas identificadas como Bacillus cereus (m363, mv86, mv81, mv75), Bacillus circulans (Fr231, m448b), Bacillus megaterium (m435), Bacillus pumilus (m354), Bacillus subtilis (m329), y Paenibacillus alvei (m321). Para probar la eficiencia de las cepas seleccionadas, se empleó la técnica de cultivo dual con 5 repeticiones de cada combinación antagonista bacteriano / cepa de A. apis, 5 para cada control y 4 medios de cultivo diferentes empleando 10 cepas del hongo de distintos orígenes geográficos. El análisis de la variancia y posterior comparación de medias LSD (Least Square Dfferences) mostró que los mejores antagonistas fueron B. subtilis (m329) y B. megaterium (m435) para el caso del medio YGPSA, mientras que para MYPGP las más eficientes fueron las cepas de B. circulans Fr 231 y m448b.

Fluorescent Pseudomonas species causing post-harvest decay of endives in Argentina

A post-harvest bacterial decay was observed on ready-to-use French endives in Argentina. Affected chicons showed browning and soft-rot of inner leaves and marginal necrosis. Physiological and biochemical tests allowed us to identify the isolates from endive as Pseudomonas fluorescens bv. III, Pseudomonas fluorescens bv. V, and Pseudomonas cichorii. Pathogenicity was verified on RTU healthy endives by inoculation with each bacterial species, and also with the mixture of the 3 strains. P. cichorii caused dark brown necrosis of the margins of outer leaves; both isolates of P. fluorescens caused browning and soft-rotting of inner leaves, while the mixture induced all the described symptoms, that were similar to those found in natural infection. Identity of bacterial isolates was confirmed by RFLP analysis of a PCR-DNA fragment amplified from the 16S rRNA gene. This is the first record of a post-harvest decay in endives in Argentina.

Tizón bacteriano de Syngonium podophyllum en la Argentina / [Bacterial leaf blight affecting Syngonium podophyllum in Argentina]

Bacterial leaf blight of Syngonium podophyllum caused by Xanthomonas campestris pv. syngonii is recorded for the first time in Argentina. The first symptom of the disease was an interveinal watersoaking of leaves, the tissues became chlorotic and finally necrotic over areas of about 4 cm. The identification of the causal microorganism was based on disease symptoms, morphological, physiological and biochemical characteristics and pathogenicity test.

Characterization of Bacillus larvae White, the causative agent of American foulbrood of honey-bees. First record of its occurrence in Argentina

American foulbrood caused by Bacillus larvae White is recorded for the first time on brood combs of Argentinian hives. The identification of the causative agent was based on disease symptomatology, morphological characters, pathogenicity tests and physiological and biochemical reactions. Studies by scanning electron microscopy showed the occurrence of large flagellar bundles of Bacillus larvae strains growing in biphasic BL medium. An electron microscope survey of the surface configuration of bacterial spores was also made.

Characterization of Bacillus larvae White, the causative agent of American foulbrood of honey-bees. First record of its occurrence in Argentina

American foulbrood caused by Bacillus larvae White is recorded for the first time on brood combs of Argentinian hives. The identification of the causative agent was based on disease symptomatology, morphological characters, pathogenicity tests and physiological and biochemical reactions. Studies by scanning electron microscopy showed the occurrence of large flagellar bundles of Bacillus larvae strains growing in biphasic BL medium. An electron microscope survey of the surface configuration of bacterial spores was also made.

Bacterial leaf spot of Zinnia elegans, a new disease in Argentina

A bacterial disease of Zinnia elegans (Compositae), caused by Xanthomonas campestris pv. zinniae is recorded for the first time in Argentina. The identification of the causal organism was based on disease symptoms, pathogenicity and morphological and physiological characteristics of the cultures isolated from material collected at La Plata, Province of Buenos Aires, Argentina.