Identification, phylogenetic analysis, and genome mining of the tetracycline-resistant Bacillus thuringiensis strain m401 reveal its potential for biotechnological and biocontrol applications.

Bacillus thuringiensis is an entomopathogen belonging to the Bacillus cereus clade. We isolated a tetracycline-resistant strain called m401, recovered it from honey, and identified it as Bacillus thuringiensis sv. kumamotoensis based on the average nucleotide identity calculations (ANIb) comparison and the analysis of the gyrB gene sequences of different B. thuringiensis serovars. Sequences with homology to virulence factors [cytK, nheA, nheB, nheC, hblA, hblB, hblC, hblD, entFM, and inhA] and tetracycline resistance genes [tet(45), tet(V), and tet(M)/tet(W)/tet(O)/tet(S) family] were identified in the bacterial chromosome. The prediction of plasmid-coding regions revealed homolog sequences to the MarR and TetR/AcrR family of transcriptional regulators, toxins, and lantipeptides. The genome mining analysis revealed 12 regions of biosynthetic gene clusters responsible for synthesizing secondary metabolites. We identified biosynthetic gene clusters coding for bacteriocins, siderophores, ribosomally synthesized post-translationally modified peptide products, and non-ribosomal peptide synthetase clusters that provide evidence for the possible use of Bt m401 as a biocontrol agent. Furthermore, Bt m401 showed high inhibition against all Paenibacillus larvae genotypes tested in vitro. In conclusion, Bt m401 owns various genes involved in different biological processes, such as transductional regulators associated with antibiotic resistance, toxins, and antimicrobial peptides with potential biotechnological and biocontrol applications.

Traceability of potential enterotoxigenic Bacillus cereus in bee-pollen samples from Argentina throughout the production process.

Bee-pollen is a functional food sold for human and animal consumption but also is a favorable microhabitat for many spore-forming bacteria. Among them, Bacillus cereus can produce several toxins and other virulence factors, causing an emetic or diarrheal syndrome after ingestion. The study involved 36 bee-pollen samples obtained from different sampling points throughout the production process (collecting, freezing, drying, and cleaning) in Argentina. Fifty isolates of B. cereus yielded 24 different fingerprint patterns with BOX and ERIC primers. Only three fingerprint patterns were maintained throughout the production process. In contrast, others were lost or incorporated during the different steps, suggesting that cross-contamination occurred as shown by differences in fingerprint patterns after freezing, drying, and cleaning steps compared to the initial collection step. Genes encoding for cereulide (ces), cytotoxin K (cytK), sphingomyelinase (sph), the components of hemolysin BL (hblA, hblB, hblC, hblD) and non-hemolytic complex (nheAB) were studied. All the isolates displayed one or more enterotoxin genes. The most frequent virulence genes detected belong to the HBL complex, being the most abundant hblA (98%), followed by hblD (64%), hblB (54%), and hblC (32%), respectively. Ten strains (20%), present at all sampling points, carried all the subunits of the HBL complex. The non-hemolytic enterotoxic complex (nheAB) was found in 48 strains (96%), while seven strains (14%) present at all sampling points showed the amplification product for sphingomyelinase (sph). One cereulide-producer was isolated at the cleaning step; this strain contained all the components for the hemolytic enterotoxin complex HBL, the NHE complex, and cytotoxin K related to the foodborne diarrhoeal syndrome. In total, 11 different virulence patterns were observed, and also a correlation between rep-fingerprint and virulence patterns. The results suggest that bee-pollen can be contaminated at any point in the production process with potential enterotoxic B. cereus strains, emphasizing the importance of hygienic processing.

Feasibility of using RFLP of PCR-amplified 16S rRNA gene(s) for rapid differentiation of isolates of aerobic spore-forming bacteria from honey.

This study aimed to assess the feasibility of using RFLP of PCR-amplified 16S rRNA gene (s) by using universal primers 27f/1492r and a combination of three restriction enzymes, AluI, CfoI, and TaqI, for a low-cost, rapid screen for a primarily differentiation of isolates of the complex of aerobic spore-forming bacteria commonly found in honey samples. The described method produced unique and distinguishable patterns to differentiate among 80 isolates belonging to 26 different species of Bacillus, Brevibacillus, Lysinibacillus, Rummeliibacillus, and Paenibacillus reported in honey and other apiarian sources.

Data associated with the characterization and presumptive identification of Bacillus and related species isolated from honey samples by using HiCrome Bacillus agar.

The dataset described in this paper provides information on the morphological features of 24 different species of the genera Bacillus, Paenibacillus, Brevibacillus, Lysinibacillus, and Rummeliibacilluswhen growing in HiCrome Bacillus agar. The species studied are common contaminants of honey. In support to the recent publication entitled "HiCrome Bacillus agar for presumptive identification of Bacillus and related species isolated from honey samples" (2), a collection of 197 bacterial isolates belonging to 24 different species of aerobic spore-forming bacteria have been screened for their colony appearance and color and any substrate color change of HiCrome Bacillus agar at 24 and 48 h of incubation. Two simple flowcharts utilizing a combination of colony and media characteristics in the chromogenic medium and a set of simple biochemical and morphological tests were developed for quick presumptive identification.

HiCrome Bacillus agar for presumptive identification of Bacillus and related species isolated from honey samples.

This study aimed to evaluate the performance of Hicrome Bacillus™ agar for isolation and rapid identification of the aerobic spore-forming bacteria most frequently found in honey samples. A collection of 197 bacterial isolates of Bacillus, Brevibacillus, Lysinibacillus, Paenibacillus, and Rummeliibacillus belonging to different species that have been reported in honey were screened for their abilities to grow and for their colony colors and medium appearance in HiCrome Bacillus agar. Also, 21 strains from culture collections were used for comparison and quality controls. A flowchart utilizing a combination of colony and media characteristics in the chromogenic medium and a set of simple biochemical and morphological tests were elaborated for quick presumptive identification. A procedure for direct isolation from honey samples was developed. In conclusion, HiCrome Bacillus agar in combination with simple microbiological tests was highly useful for rapid and reliable identification of most Bacillus, Brevibacillus, Lysinibacillus and Paenibacillus species commonly found in honey samples facilitating isolation from polymicrobial honey.

Draft Genome Sequence of Bacillus thuringiensis Strain m401, Isolated from Honey in Argentina.

We report here the 6,092,003-bp draft genome sequence of Bacillus thuringiensis strain m401, a tetracycline-resistant isolate recovered from honey. The isolate contained three plasmids of 8,307 bp, 9,934 bp, and 69,561 bp and a tetracycline resistance gene with high homology to tet45 in a contig of 236,180 bp.

A case of intoxication due to a highly cytotoxic Bacillus cereus strain isolated from cooked chicken.

Outbreaks of Bacillus cereus infection/intoxication are not commonly reported because symptoms are often mild, and the disease is self-limiting. However, hypervirulent strains increase health risks. We report a case, which occurred in Argentina, of severe food poisoning illness on a healthy adult woman associated to B. cereus strain MVL2011. The studied strain was highly cytotoxic, showed high ability to detach Caco-2 cells and was positive for the hblA, hblB, and hblC genes of the hbl complex, bceT, entS and ces. As it is considered that B. cereus emetic cluster evolved from a panmictic population of diarrheal strains, B. cereus MVL2011 could constitute an intermediate strain between diarrheal and emetic strains.

[Agrobacterium rubi strains from blueberry plants are highly diverse]. / Diversidad de cepas de Agrobacterium rubi aisladas de arándanos.

The diversity of a collection of Agrobacterium rubi strains isolated from blueberries from different regions of Argentina was studied by conventional microbiological tests and molecular techniques. Results from biochemical and physiological reactions, as well as from rep-PCR and RFLP analysis of PCR-amplified 23S rDNA showed high phenotypic and genotypic intraspecific variation.

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.

Diversidad de cepas de Agrobacterium rubi aisladas de arándanos / Agrobacterium rubi strains from blueberry plants are highly diverse

Se estudió la diversidad de una colección de cepas de Agrobacterium rubi aisladas de arándanos provenientes de distintas regiones de la República Argentina estableciendo su grado de heterogeneidad mediante pruebas microbiológicas clásicas y técnicas de biología molecular. Los resultados obtenidos en las pruebas bioquímicas y fisiológicas, así como mediante rep-PCR y RFLP del gen 23S ADNr, demostraron una alta variabilidad intraespecífica, tanto fenotípica como genotípica

The diversity of a collection of Agrobacterium rubi strains isolated from blueberries from different regions of Argentina was studied by conventional microbiological tests and molecular techniques. Results from biochemical and physiological reactions, as well as from rep-PCR and RFLP analysis of PCR-amplified 23S rDNA showed high phenotypic and genotypic intraspecific variation

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 / [Evaluation of the Epsilometer (Etest) method for the detection of tetracycline susceptibility in Paenibacillus larvae, the causal agent of American foulbrood disease of honeybees].

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 Iso- Sensitest 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 errors) when MYPGP was tested. In conclusion, the Etest could be a rapid and reliable method for testing MIC values of tetracycline in P. larvae only when used in combination with Iso-Sensitest agar. Nevertheless, these results should be confirmed with future studies involving a larger number of isolates.

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 Dulbecco's modified Eagle's 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.

[Evaluation of the Epsilometer (Etest) method for the detection of tetracycline susceptibility in Paenibacillus larvae, the causal agent of American foulbrood disease of honeybees]. / 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 Iso- Sensitest 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 errors) when MYPGP was tested. In conclusion, the Etest could be a rapid and reliable method for testing MIC values of tetracycline in P. larvae only when used in combination with Iso-Sensitest agar. Nevertheless, these results should be confirmed with future studies involving a larger number of isolates.

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

El género Agrobacterium incluye especies ftopatógenas que inducen la formación de agallas en el cuello o la proliferación de raíces en cabellera en más de 600 especies de dicotiledóneas, y especies no patógenas cuyo hábitat natural es el suelo. Como no es posible erradicar a las especies patógenas y habida cuenta de que más del 80 % de las infecciones puede provenir de viveros, es importante evitar la diseminación de la enfermedad. Por ello, el objetivo de este trabajo ha sido desarrollar técnicas sensibles y precisas que, aisladamente o combinadas, permitan detectar la presencia de especies y biovares de Agrobacterium a partir de muestras de material vegetal, suelo y agua. Se comprobó que con la estrategia combinada de realizar aislamientos en los medios semiselectivos D1, D1-M y YEM-RCT; PCR multiplex sobre el gen 23S ADNr; PCR específca sobre los genes virC1 y virC2 y bioensayos en plántulas de pimiento cv. California Wonder y en hojas cortadas de kalanchoe, se reduce la posibilidad de obtener falsos negativos y/o falsos positivos. Por lo expuesto, esta combinación de técnicas constituye una herramienta adecuada para el diagnóstico de cepas patógenas de Agrobacterium a partir de distintos tipos de muestras.

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 diffcult 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 identifcation techniques by using a set of semi-selective and differential culture media in combination with a specifc 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, specifc 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.

[Methods for the detection of Agrobacterium from plant, soil and water samples]. / Métodos para la detección de Agrobacterium a partir de muestras de material vegetal, suelo y agua.

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.

Effectiveness of tilmicosin against Paenibacillus larvae, the causal agent of American Foulbrood disease of honeybees.

American Foulbrood (AFB) of honeybees (Apis mellifera L.), caused by the Gram-positive bacterium Paenibacillus larvae is one of the most serious diseases affecting the larval and pupal stages of honeybees (A. mellifera L.). The aim of the present work was to asses the response of 23 strains of P. larvae from diverse geographical origins to tilmicosin, a macrolide antibiotic developed for exclusive use in veterinary medicine, by means of the minimal inhibitory concentration (MIC) and the agar diffusion test (ADT). All the strains tested were highly susceptible to tilmicosin with MIC values ranging between 0.0625 and 0.5 microg ml(-1), and with MIC(50) and MIC(90) values of 0.250 microg ml(-1). The ADT tests results for 23 P. larvae strains tested showed that all were susceptible to tilmicosin with inhibition zones around 15 microg tilmicosin disks ranging between 21 and 50mm in diameter. Oral acute toxicity of tilmicosin was evaluated and the LD(50) values obtained demonstrated that it was virtually non-toxic for adult bees and also resulted non-toxic for larvae when compared with the normal brood mortality. Dosage of 1000 mg a.i. of tilmicosin applied in a 55 g candy resulted in a total suppression of AFB clinical signs in honeybee colonies 60 days after initial treatment. To our knowledge, this is the first report of the effectiveness of tilmicosin against P. larvae both in vitro and in vivo.

Evidence for plasmid-mediated tetracycline resistance in Paenibacillus larvae, the causal agent of American Foulbrood (AFB) disease in honeybees.

Paenibacillus larvae is the causal agent of American Foulbrood (AFB) disease, the most virulent bacterial disease of honeybee (Apis mellifera L.) brood. Oxytetracycline is the main antibiotic used for prevention and control of AFB. Using the polymerase chain reaction, isolates were screened for the presence of the tetracycline resistance tet(K) and tet(L) determinants. Four isolates (5%), which correlated with the Tc-resistant phenotypes, were found to carry the tet(K) determinant, whereas none carried the tet(L) determinant. P. larvae cells were also screened for the presence of extrachromosomal DNA and evidence obtained that tetracycline resistance is plasmid-encoded. A few P. larvae isolates were found to be able to transfer the tet(K) determinant to Bacillus subtilis, suggesting that a conjugation mechanism may be involved in the transfer of the tetracycline-resistant phenotype. Minimum inhibitory concentrations to tetracycline were determined for 75 isolates of P. larvae from different geographical origins and found to range between 0.062 and 128 microg tetracyclineml(-1), with MIC(50) and MIC(90) values of 1 and 4, respectively. According to results from P. larvae populations, isolates could be considered as susceptible when their MICs were <4, intermediate for MICs values 4-8 and resistant for MICs > or = 16. To our knowledge, this is the first report of Tc(r)Paenibacillus species carrying a tet(K) gene, and also the first record of P. larvae strains carrying tet(K) determinants and its correlation with the presence of extrachromosomal DNA.

Phenotypic and genotypic diversity of Bacillus cereus isolates recovered from honey.

Of 433 honey samples collected in Argentina, 114 (27%) yielded Bacillus cereus isolates (27%) and 60 (14%) yielded other species of Bacillus. The Argentinian B. cereus isolates were compared with isolates recovered from honey from other countries (n=16) and with strain ATCC 11718. The 133 B. cereus isolates and the ATCC strain were characterized using rep-PCR fingerprinting with primers BOX, REP and ERIC, restriction fragment length polymorphism analysis of a 16S rRNA gene fragment (16S rRNA PCR/RFLP), and morphological and biochemical tests. There was a high degree of diversity, both phenotypic and genotypic among the isolates of B. cereus. Our results demonstrate the usefulness of rep-PCR genomic fingerprinting for characterizing populations of B. cereus. According to our knowledge, this is the first report of phenotypic and genotypic characterization of B. cereus isolates from honey.

Inhibition of the growth of Paenibacillus larvae, the causal agent of American foulbrood of honeybees, by selected strains of aerobic spore-forming bacteria isolated from apiarian sources.

The bacterium Paenibacillus larvae, the causative agent of American foulbrood disease of honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of antagonic aerobic spore-forming bacteria isolated from honey samples and other apiarian sources were evaluated. Each isolate was screened against one strain of Paenibacillus larvae (ATCC 9545) by using a perpendicular streak technique. Ten randomly selected bacterial strains from the group that showed the best antagonistic effect to P. larvae ATCC 9545 were selected for further study. These were identified as Bacillus subtilis (m351), B. pumilus (m350), B. licheniformis (m347), B. cereus (mv33), B. cereus (m387), B. cereus (m6c), B. megaterium (m404), Brevibacillus laterosporus (BLAT169), B. laterosporus (BLAT170), and B. laterosporus (BLAT171). The antagonistic strains were tested against 17 P. larvae strains from different geographical origins by means of a spot test in wells. The analysis of variance and posterior comparison of means by Tukey method (P < 0.01) showed that the best antagonists were B. megaterium (m404), B. licheniformis (m347), B. cereus (m6c), B. cereus (mv33), and B. cereus (m387).

In vitro and in vivo susceptibility of the honeybee bacterial pathogen Paenibacillus larvae subsp. larvae to the antibiotic tylosin.

The minimal inhibitory concentrations (MICs) of tylosin were determined to 67 strains of Paenibacillus larvae subsp. larvae, the causal agent of American Foulbrood (AFB) disease, from different geographical origins. MIC values obtained ranged from 0.0078 to 0.5 microg/ml. These very low values imply that no resistance to tylosin was found in any isolate of the Foulbrood pathogen. The measurement of diseased larvae with AFB-clinical symptoms in three different field studies demonstrated that tylosin treatment could be effective in vivo. No negative effects in colonies were noted at any dosage rates or forms of application. These studies demonstrate that tylosin, as tartrate, can be used to treat AFB in honeybee colonies.