R ESULTS
1. Development of PCR tools targeted to AMP genes for screening field samples and culture collections of Bacillus
2.3 Evaluation of selection and enrichment strategies
Figure 2.14. Kinetics of survival B. subtilis strains RGAF32 ( ), EPS2004 ( ), and UMAF6639 ( ), B. megaterium RGAF51 ( ), compared to the plant associated bacteria Pseudomonas fluorescens EPS62e ( ) and Pantoea agglomerans EPS125 ( ) after thermal treatment at 80 ºC at different exposure times.
Figure 2.15. Thermal sensitivity of B. subtilis strains RGAF32 ( ), EPS2004 ( ), and UMAF6639 ( ), B. megaterium RGAF51 ( ), compared to the plant associated bacteria Pseudomonas fluorescens EPS62e ( ) and Pantoea agglomerans EPS125 ( ).
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Total culturable bacteria populations and population of amended Bacillus RGAF51 Rif+ at different initial concentrations (104, 105, 106 and 107 cfu/ml) were determined for each step of the procedure (untreated, after thermal treatment, at the end of the enrichment) (Fig. 2.16). In the extracts without addition of Bacillus, the population of total culturable bacteria decreased to undetectable levels after thermal treatment at both times of exposure tested with a reduction higher than 4 logs. Besides, these populations were not recovered after the enrichment stage in none of the dilutions assessed. In contrast, the population levels of Bacillus RGAF51 Rif+, decreased to only around 2 logs after the thermal treatment and differences were not observed between the two thermal exposure times.
However, significant differences in populations of Bacillus RGAF51 Rif+ were observed depending on the dilution conditions during the enrichment step.
Recoveries of Bacillus did not differ when sample extracts were amended with high or low initial concentrations (107 and 106 cfu/ml) achieving recovery values of 5x106 cfu/ml in both cases. However, the recovery of Bacillus added at low initial concentrations (105 and 104 cfu/ml) was affected by the dilution during enrichment, with recovery values around 106 cfu/ml using dilution 1:100, but of only 103 cfu/ml with dilution 1:1000, independently of the thermal treatment applied. Moreover, Bacillus added at low initial concentrations were not recovered when the sample was submitted to a thermal treatment at 80 ºC for 20 min and to an enrichment in 1:1000 dilution.
2.4.2 Evaluation
The selective enrichment method was evaluated in two assays consisting of the addition of a Bacillus strain to mixture of Gram-negative strains (artificial microcosm) and field extracts. The addition of a Bacillus strain in a mixture of Gram-negative bacteria was performed in order to compare the survival of heat-sensitive bacteria, such as chosen Gram-negative strains with the thermoresistant Bacillus strain. Population levels of total culturable bacteria and of a Bacillus spp. were determined for each step of the procedure (initial untreated, after thermal treatment, at the end of the enrichment stage) (upper panel Fig. 2.17).
Figure 2.16. Population levels of total culturable bacteria and of B. megaterium RGAF51Rif+ from rhizosphere extract at different stages of the selective enrichment method.Untreated (U),after thermaltreatment (T) and after thermal treatment and enrichment (TE). Different initial concentrations of RGAF51Rif+ were used (0 (■), 104 (■), 105 (■), 106 (■) and 107 cfu/ml (■)). Different extract dilution was used before the enrichment step (diluted 1:100 and diluted 1:1000). Different exposure time of thermal treatment were used 20 min and 10 min. Total culturable bacteria (■) were determined in LB agar whereas culturable rifampicin resistant bacteria (■, ■,■,■) were determined on LB agar supplemented with rifampicin (100 mg/l). The minimum detection level was 1.7 log10 cfu/ml. nd, not detected.
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Figure 2.17. Population of total culturable bacteria (■) and B. megaterium RGAF51 Rif+ (■) from artificial microcosm (upper panel) and natural plant microbiota (Lower pannel). Populations were determined in different stages of the selective enrichment method: Untreated (U), after thermal treatment (T) and after thermal treatment and enrichment (TE). Different initial concentrations of RGAF51 Rif+ were used (0, 103, 104, and 106 cfu/ml). Total culturable bacteria were determined in LB agar whereas culturable rifampicin resistant bacteria were determined on LB agar supplemented with rifampicin (100 mg/l). The minimum detection level was 1.7 log10 cfu/ml.
Confidence intervals over the bars correspond to three replicates. (*) Indicates that autochthonous Bacillus grew into one out of the three replicates. nd indicates non-detected colonies.
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In the mixture of Gram-negative strains without addition of the Bacillus strain, the total bacterial culturable population decreased 6 logs, until undetectable values, after the thermal treatment, and bacteria were not recovered after the enrichment. When the rifampicin resistant Bacillus RGAF51 Rif+ was added a reduction in the total culturable bacteria after the thermal treatment was also observed, although this reduction was related to the initial population of Bacillus added (1x103, 1x104 and 1x106 cfu/ml).
The rifampicin resistant Bacillus population did not significantly changed after the thermal treatment and coincided with the total population of culturable bacteria. After the enrichment step Bacillus populations increased to values above 107 cfu/ml, independently of the initial populations inoculated.
Similar results were obtained in the assay performed with field sample extracts. Total culturable bacteria populations in the plant microbiota extract without the addition of Bacillus decreased to undetectable values after the thermal treatment (lower panel Fig. 2.17). Only in one of the three replicates a residual population was recovered after enrichment and this was due to presence of autochthonous Bacillus, which wasproved to be susceptible after isolation to the antibiotic. These results were confirmed by PCR with primers for Bacillus targeted to the 16S rDNA gene (Fig. 2.18).
Field extracts without addition of Bacillus did not gave amplification for the 16S rDNA gene nor for the antimicrobial peptide genes (bmyB, fenD, ituC, srfAA, bacA and spaS), neither in the untreated samples nor after the thermal treatment, except for the above mentioned sample where autochthonous Bacillus were detected.
In contrast, all the samples amended with Bacillus RGAF 51 Rif+ showed positive amplification for all primer pairs except in the samples amended to a final concentration of Bacillus of 104 and 103, just after the thermal treatment. Moreover, the intensity of bands was proportional to the initial concentration of Bacillus inoculated.
Figure 2.18. PCR detection of Bacillus by means of general primers (16S rDNA) in natural plant extract and water controls with or without amendment of B. subtilis RGAF51Rif+ at different concentrations and in different stages of the selective enrichment method. Initial concentrations of RGAF51Rif+ were 0, 103, 104, and 106 cfu/ml. M, 1Kb Plus DNA Ladder (Invitrogen).
2.5 Comparison of the standard and selective enrichment methods