Ana Claudia López

Differentiation of Paenibacillus larvae subsp. larvae, the Cause of American Foulbrood of Honeybees, by Using PCR and Restriction Fragment Analysis of Genes Encoding 16S rRNA

ABSTRACT A rapid procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American foulbrood (AFB) disease of honeybees (Apis mellifera L.), based on PCR and restriction fragment analysis of the 16S rRNA genes (rDNA) is described. Eighty-six bacterial strains belonging to 39 species of the genera Paenibacillus, Bacillus, Brevibacillus, and Virgibacillus were characterized. Amplified rDNA was digested with seven restriction endonucleases. The combined data from restriction analysis enabled us to distinguish 35 profiles. Cluster analysis revealed that P. larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens formed a group with about 90% similarity; however, the P. larvae subsp. larvae restriction fragment length polymorphism pattern produced by endonuclease HaeIII was found to be unique and distinguishable among other closely related bacteria. This pattern was associated with DNA extracted directly from honeybee brood samples showing positive AFB clinical signs that yielded the restriction profile characteristic of P. larvae subsp. larvae, while no amplification product was obtained from healthy larvae. The method described here is particularly useful because of the short time required to carry it out and because it allows the differentiation of P. larvae subsp. larvae-infected larvae from all other species found in apiarian sources.

Molecular epidemiology of Paenibacillus larvae larvae and incidence of American foulbrood in Argentinean honeys from Buenos Aires province

SUMMARY Paenibacillus larvae larvae, the causative agent of American foulbrood disease of honey bee larvae occurs throughout the world and is found in all beekeeping areas of Argentina. Microbiological analysis of 394 honey samples obtained from bee hives from Buenos Aires province (Argentina) from three years of sampling (1999–2001) yielded 219 positive cases (55.6%). The incidence of P l. larvae infected honey samples for 1999 was 68.1% (n = 160), for 2000 47.1% (n = 102), and 46.2% for 2001 (n = 132). The mean values of spore contamination for the three-year study showed a continuous reduction, probably due to good practices of disease management by beekeepers by breeding bees for hygienic behaviour and reduction of antibiotic treatments for control of AFB. P. l. larvae populations isolated from honey were characterized on the basis of DNA fingerprints using the repetitive-sequence-based polymerase chain reaction technique (rep-PCR) with BOX- and REP- sequence- specific primers. Four distinctive patterns, named A, B, C, and D, were distinguishable among the isolates. Genotype D was not observed in previous studies; this finding could be correlated with a new introduction of the disease in Argentina since 1997 when only three genotypes (A, B, and C) were confirmed. The rep-PCR fingerprint patterns obtained were compared with the patterns generated by a world-wide collection of P. l. larvae strains. The same 4 genotypes patterns were found within a collection of strains from 18 different countries of the world. It is important to point out that pattern C was only found in Argentina and in one sample from Uruguay located in the border line, suggesting that genotype C could have been derived from genotype A and disseminated to Uruguay from Argentina. These findings support the hypothesis that American foulbrood disease is exposed to a limited selective pressure from climatic and environmental sources.

A PCR‐based method that permits specific detection of Paenibacillus larvae subsp. larvae, the cause of American Foulbrood of honey bees, at the subspecies level

Aims:  A reliable procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American Foulbrood disease of honey bees (Apis mellifera L.) based on the polymerase chain reaction (PCR) and subspecies – specific primers is described.

Differentiation of Ascosphaera apis isolates by rep-PCR fingerprinting and determination of chalkbrood incidence in Argentinean honey samples

SUMMARY Ascosphaera apis, the causative agent of chalkbrood disease in honey bee larvae occurs throughout the world and is found in many beekeeping areas of Argentina. Microbiological analysis of 394 honey samples from Buenos Aires province from three years of sampling (1999–2001) yielded 51 positive cases (13% incidence). Eighty-four isolates of A. apis from Argentina and Chile isolated from diseased larvae and/or honey samples were characterized on the basis of DNA fingerprints using the repetitive-sequence-based polymerase chain reaction technique (rep-PCR) with BOX, REP, and ERIC sequence-specific primers. Computer-assisted analysis of combined fingerprints distinguished six groups of patterns, designated A, B, C, D, E and F. Pattern C was the most prevalent and suggests a limited diversity in the populations of A. apis from Argentina and Chile. The results demonstrated the usefulness of rep-PCR genomic fingerprinting to characterize populations of A. apis. In addition, a simple and efficient protocol for the extraction of total fungal genomic DNA was developed that was appropriate for simultaneous and cost-efficient processing of many samples.

First Report of Leaf Spot Disease of Maize Caused by Pantoea ananatis in Argentina

From 2007 to 2008, an uncharacterized disease of maize (Zea mays L.) was observed in commercial fields of Laguna Blanca, Formosa, Argentina and from different fields of Santa Fe and Catamarca provinces of Argentina. Symptoms included light-colored necrotic streaks on leaves and tan or white irregular blotches that sometimes were surrounded by reddish purple-to-dark brown margins. Severity of symptoms varied greatly from one field to another. Abundant bacterial streaming was observed from lesions when examined at ×150. Gram-negative, facultatively anaerobic bacteria were consistently isolated from lesions. These formed light yellow-to-orange, glistening, convex colonies on yeast dextrose calcium carbonate agar incubated at 30°C. Ten isolates from ten different symptomatic plants were selected for further study. All isolates were motile, induced a hypersensitive response in tobacco plants, and were oxidase negative. Colonies developed at 37°C. Physiological and biochemical characterization with the API 20E test strips and database (bioMerieux, Buenos Aires, Argentina) showed that the strains belonged to the genus Pantoea. All strains were positive for β-galactosidase, utilized citrate and tartrate, and produced acid from d-glucose, d-mannitol, d-melibiose, l-arabinose, sucrose, meso-inositol, glycerol, d-sorbitol, and amygdalin. All were negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophane deaminase, H2S production, urease, and reduction of nitrate to nitrite. Variable results were obtained for indole, gelatinase, and l-rhamnose. Their identity was confirmed by sequencing the 16S rRNA gene strain F327 (GenBank Accession No. GU068363). A BlastN search of GenBank revealed 99% nt identity with strains LMG 20103 (AF364847.1), LMG 20105 (AF364845.1), and LMG 2665 (FJ611815.1) of Pantoea ananatis. Pathogenicity was verified on Z. mays (EM 6079 HX, Dow Morgan) by injection-infiltration of bacterial suspensions at 105 CFU/ml. Controls were infiltrated with sterile distilled water. Plants were kept at 26 ± 3°C in a greenhouse. Symptoms were first detected 15 to 17 days after inoculation and then lesions expanded to resemble natural infections within 30 days. Bacteria were reisolated and the original and reisolated strains were compared by using repetitive sequence-based (rep)-PCR with ERIC primers (1) and fingerprints of the reisolated strains were identical to those of the original strains, thereby fulfilling Kochs postulates. No lesions were observed on controls. Known strains of P. stewartii from the United States (SW2, DC400, DC441, and DC283) were also tested for comparison. On the basis of sequencing data, pathogenicity, and physiological tests, the pathogen was identified as P. ananatis (4). To our knowledge, this is the first report of P. ananatis causing a disease of maize in Argentina, although a similar disease has been reported in Brazil (2) and Mexico (3). References: (1) F. J. Louws et al. Appl. Environ. Microbiol. 60:2286, 1994. (2) L. D. Paccola-Meirelles et al. J. Phytopathol. 149:275, 2001. (3) R. Pérez-y-Terrón et al. Australas. Plant Dis. Notes 4:96, 2009. (4) N. W. Schaad et al., eds. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001.

Tetracycline-resistance encoding plasmids from Paenibacillus larvae , the causal agent of American foulbrood disease, isolated from commercial honeys

Paenibacillus larvae, the causal agent of American foulbrood disease in honeybees, acquires tetracycline-resistance via native plasmids carrying known tetracycline-resistance determinants. From three P. larvae tetracycline-resistant strains isolated from honeys, 5-kb-circular plasmids with almost identical sequences, designated pPL373 in strain PL373, pPL374 in strain PL374, and pPL395 in strain PL395, were isolated. These plasmids were highly similar (99%) to small tetracycline-encoding plasmids (pMA67, pBHS24, pBSDMV46A, pDMV2, pSU1, pAST4, and pLS55) that replicate by the rolling circle mechanism. Nucleotide sequences comparisons showed that pPL373, pPL374, and pPL395 mainly differed from the previously reported P. larvae plasmid pMA67 in the oriT region and mob genes. These differences suggest alternative mobilization and/or conjugation capacities. Plasmids pPL373, pPL374, and pPL395 were individually transferred by electroporation and stably maintained in tetracycline-susceptible P. larvae NRRL B-14154, in which they autonomously replicated. The presence of nearly identical plasmids in five different genera of gram-positive bacteria, i.e., Bhargavaea, Bacillus, Lactobacillus, Paenibacillus, and Sporosarcina, inhabiting diverse ecological niches provides further evidence of the genetic transfer of tetracycline resistance among environmental bacteria from soils, food, and marine habitats and from pathogenic bacteria such as P. larvae.

In vitro interaction between Bacillus megaterium strains and Caco-2 cells

To further our understanding of the virulence potential of Bacillus megaterium strains, cell association and invasion assays were conducted in vitro by infecting human enterocytes (Caco-2 cells) with 53 strains of this bacterium isolated from honey. Two series of experiments were performed: (i) necrosis and cell detachment assays with the supernatants of bacterial culture filtrates from 16-h cultures and (ii) adhesion/invasion assays in which cultured enterocytes incubated with bacteria from 3-h cultures were resuspended in Dulbeccos modified Eagles medium and chloramphenicol. The detachment of Caco-2 cells was evaluated by staining the cells with crystal violet. Necrosis was assessed by fluorescence microscopy of cells labeled with propidium iodide. Association (adhesion plus invasion) was determined by plate counts and invasion in an aminoglycoside protection assay. The results showed that spent culture supernatants detached and necrotized Caco-2 cells in a strain-dependent manner. Seven out of 53 B. megaterium filtered culture supernatants caused complete cell detachment. Suspensions of these same bacterial strains adhered and invaded enterocytes in 2-h infection experiments. To our knowledge, this is the first report on the interaction between B. megaterium and intestinal epithelial Caco-2 cells.

Métodos para la detección de Agrobacterium a partir de muestras de material vegetal, suelo y agua

Methods for the detection of Agrobacterium from plant, soil and water samples. The genus Agrobacterium includes phytopathogenic bacteria that induce the development of root crown galls and/or aerial galls at the base of the stem or hairy roots on more than 600 species of plants belonging to 90 dicotyledonous families and non-pathogenic species. These bacteria being natural soil inhabitants are particularly difficult to eradicate, which is a problem in nurseries where more than 80% of infections occur. Since early detection is crucial to avoid the inadvertent spread of the disease, the aim of this work was to develop sensitive and precise identification techniques by using a set of semi-selective and differential culture media in combination with a specific PCR to amplify a partial sequence derived from the virC operon, as well as a multiplex PCR on the basis of 23SrDNA sequences, and biological assays to identify and differentiate species and biovars of Agrobacterium obtained either from soil, water or plant samples. The combination of the different assays allowed us to reduce the number of false positive and negative results from bacteria isolated from any of the three types of samples. Therefore, the combination of multiplex PCR, specific PCR, isolations in semi-selective D1, D1-M and YEM-RCT media combined with bioassays on cut leaves of Kalanchoe and seedlings of California Wonder pepper cultivar constitute an accurate tool to detect species and biovars of Agrobacterium for diagnostic purposes.

Bacillus and Brevibacillus strains as potential antagonists of Paenibacillus larvae and Ascosphaera apis.

Species of Bacillus and Brevibacillus associated with honey bees are interesting sources of bioactive compounds with potential uses beyond the field of apiculture. Most Bacillus species and related genera produce a broad range of antimicrobial compounds, with activity against bacteria and fungi that include peptides, lipopeptides, bacteriocins, and bacteriocin-like inhibitory substances. By using biological tools, we evaluated the antagonistic activity of 34 bacterial strains against Paenibacillus larvae and Ascosphaera apis, the causal agents of American Foulbrood and Chalkbrood diseases of honey bee larvae, respectively. Data reveal that the antagonistic response was strain-specific, species-specific, and also medium-dependent. By using molecular tools, we investigated the distribution of antimicrobial peptide genes in the antagonist strains. The presence of homologous sequences to nine genes encoding for the synthesis of the antimicrobial peptides bacillomycin L (bmyB), fengycin (fenD), bacilysin (bacA), subtilin (spaS), iturin A (ituD, lpa-14; ituC), and surfactin (sfp; srfAA) was assayed by PCR. The distribution and frequency of these genes within the bacterial antagonists were also variable and strain-dependent, being the most common surfactins (srfAA = 44% and lpa-14 = 38%), iturins (ituD = 47%), and bacilysin (bacA = 32%). Moreover, a positive correlation between presence of antimicrobial peptide genes and antagonism was found taking into account that 85% of the antagonists had at least one of the antimicrobial peptide genes. We also identified those antagonists active against different P. larvae genotypes. To our knowledge, this is the first study of the association between the presence of homologous sequences of antimicrobial peptide genes and antagonism against P. larvae and A. apis strains. Cepas de Bacillus and Brevibacillus como antagonistas potenciales de Paenibacillus larvae y Ascosphaera apis. Las especies de Bacillus y Brevibacillus asociadas con abejas melíferas son una fuente interesante de compuestos bioactivos con usos potenciales más allá del campo de la apicultura. La mayoría de las especies de Bacillus producen una amplia gama de compuestos antimicrobianos, con actividad contra bacterias y hongos que incluye péptidos, lipopéptidos, bacteriocinas y sustancias inhibidoras similares a bacteriocinas (BLIS). Con el objeto de buscar alternativas naturales para el control de loque americana y cría yesificada, se emplearon herramientas biológicas para evaluar la actividad antagónica de 34 cepas bacterianas contra cepas de Paenibacillus larvae y de Ascosphaera apis, agentes causales de estas enfermedades. Los resultados obtenidos mostraron que la respuesta antagónica fue medio-dependiente y cepa-dependiente. Se analizó por PCR la presencia de secuencias homólogas a 9 genes relacionados con la síntesis de péptidos antimicrobianos: bacilomicina L (bmyB), fengicina (fenD), bacilicina (bacA), subtilina (spaS), iturina A (ituD, lpa-14; ituC), y surfactina (sfp; srfAA). La distribución y frecuencia de estos genes en las cepas antagonistas también resultó variable y cepa-dependiente, siendo los más comunes surfactinas (srfAA = 44% y lpa-14 = 38%), iturina A (ituD = 47%) y bacilicina (bacA = 32%). Se identificaron antagonistas bacterianos frente a distintos genotipos de P. larvae y se encontró una correlación positiva entre la presencia de genes vinculados con la producción de péptidos antimicrobianos y la inhibición del desarrollo de P. larvae. Este trabajo constituye el primer estudio de asociaciones entre presencia de estos genes y el antagonismo frente a P. larvae y A. apis.

Evaluación del método epsilométrico Etest para la determinación de la sensibilidad a tetraciclina en Paenibacillus larvae, agente causal de la loque americana de las abejas

American foulbrood (AFB) is a bacterial disease caused by the spore-forming, grampositive bacterium Paenibacillus larvae, which affects honeybee broods worldwide. The aim of this work was to compare the Epsilometer test (Etest) to the agar dilution method for testing a collection of 22 P. larvae strains to tetracycline by using MYPGP and IsoSensitest agars. Results showed that a categorical agreement of 100% was found when using Iso-Sensitest, while a categorical agreement of 86.36% was found (with 3 minor