Antibacterial activity against enterovirulent Escherichia coli strains from Bacillus amyloliquefaciens B31 and Bacillus subtilis subsp. subtilis C4: MALDI-TOF MS profiling and MALDI TOF/TOF MS structural analysis on lipopeptides mixtures.

In this work, two Bacillus strains isolated from honey (Bacillus subtilis subsp. subtilis C4; access code HQ828992) and from a waste of an artisanal tannery (Bacillus amyloliquefaciens B31; access code KP893752) were evaluated in order to determine their antibacterial activity against five enteropathogenic Escherichia coli strains. The number of viable cultivable cells of the different strains of E. coli analyzed was determined by plate count. The crude cell-free supernatants of both Bacillus strains exerted anti-E. coli activities, whereas only the lipopeptide fraction of B31 had significant E. coli inhibition. The lipopeptides produced by the Bacillus were analyzed using matrix-assisted laser desorption-ionization mass spectrometry (MALDI MS). The analysis was conducted combining the profiles (fingerprints) of the lipopeptides mixture and the individual lipopeptide fragmentation (tandem mass spectrometry [MS/MS] mode), both obtained from the same lipopeptides mixture sample, for higher output. Data obtained from C4 and B31 revealed that surfactin homologues were the most abundant lipopeptides produced by both strains studied. Additionally, kurstakin, iturin, and fengycin homologues were detected. Using the MS/MS mode, it was demonstrated that isobar compounds belonging to different families were produced by each Bacillus strain (e.g., C-16 bacillomycin D was detected in B31 samples, meanwhile C-15 iturin C was detected in C4). MS/MS analysis contributed with relevant information about the type of lipopeptides synthesized by Bacillus strains studied in this work.

Antibacterial activity of Bacillus licheniformis B6 against viability and biofilm formation of foodborne pathogens of health importance.

We studied a strain of Bacillus isolated from an artisanal tannery in Salta, Argentina. It was identified as Bacillus licheniformis B6 by 16 S phylogenetic analysis and MALDI TOF (GenBank accession code No. KP776730). The synthesis of lipopeptides by B6 and their antibacterial activity against clinical pathogenic strains was analyzed both in the cell-free supernatant (CFS) and in the crude fraction of lipopeptides (LF). Overall, the CFS did not significantly reduce the viability of the studied strains (Staphylococcus aureus 269 and ATCC 43,300, Escherichia coli 4591 and 25,922, Klebsiella sp. 1087 and 1101). However, LF at 9 mg/mL reduced the viability of those pathogenic strains by 2 and 3 log orders compared to those of the control. When the effects of LF and ampicillin were compared, they showed different sensitivity against pathogenic strains. For example, E. coli 4591 was the strain most resistant to ampicillin, requiring 250 mg/mL of antibiotic to achieve the same inhibitory effect as 9 mg/mL of B6 LF. SEM observations of the effect of LF on biofilm formation by E. coli 4591 and Klebsiella sp. 1087 clearly showed that biofilm structures were destabilized, these strains turning into weak biofilm formers. Signals in the CFS and LF corresponding to kurstakin and iturin were identified by MALDI TOF. Interestingly, surfactin was detected, rather than lichenysin, the expected lipopeptide in B. licheniformis species. Signals of bacitracin and fengycins were also found, the latter with a higher number of homologues and relative intensity in the LF than the other lipopeptides. These results show that the lipopeptides synthesized by B. licheniformis B6 have both potential antibacterial and anti-biofilm activity against pathogenic bacteria of health importance.

Lactobacillus salivarius A3iob Reduces the Incidence of Varroa destructor and Nosema Spp. in Commercial Apiaries Located in the Northwest of Argentina.

Lactobacillus salivarius A3iob was administered to productive colonies belonging to commercial apiaries of small beekeepers (around 30-50 hives each one), from four departments of the province of Jujuy (Argentina): Yala, Tilquiza, El Carmen, and Los Alisos. The incidence of Varroa destructor and Nosema spp., before and after winter, was monitored during 2 years of study (2014-2015). Depending on the geographical location of each apiary and the application time, a monthly dose of the bacteria (105 CFU/mL) reduced the levels of varroasis between 50 and 80%. Interestingly, L. salivarius A3iob cells remitted the percentage of the mites to undetectable values in an apiary treated with flumethrin (at Yala, Yungas region).On the other hand, the spore levels of Nosema spp. in the lactobacilli-treated colonies also depended on the apiary and the year of application, but a significant decrease was mainly observed in the post-winter period. However, at Rivera (El Carmen's department), no significant changes were detected in both parameters.These results obtained after 2 years of work suggest that delivering L. salivarius A3iob cells to the bee colonies can become a new eco-friendly tool to cooperate with the control of these bees' pests.

Aphicidal activity of Bacillus amyloliquefaciens strains in the peach-potato aphid (Myzus persicae).

Myzus persicae Sulzer (Hemiptera: Aphididae), is a generalist cosmopolitan insect that infests more than 400 plant species of 40 different families and is one of the major pests infesting potato crops. It causes direct damage and also spread plant viruses. The intensive use of synthetic insecticide to control aphids has led to resistant populations. Therefore, there is a need to develop biopesticides for effective control that minimizes environmental hazards. The bacteria Bacillus amyloliquefaciens is recognized as a producer of a variety of bioactive compounds. The aim here was to evaluate the aphicidal effect of B. amyloliquefaciens strains, CBMDDrag3, PGPBacCA2, and CBMDLO3, and their metabolites on the mortality and fecundity of M. persicae. Cells suspensions, heat-killed cell suspensions, cell-free supernatants, or isolated lipopeptide fractions from B. amyloliquefaciens strains were offered to aphids through artificial diets. The isolated lipopeptide fractions composed mainly of kurstakins, surfactins, iturins, and fengycins, when were administrated through diets, had no aphicidal effect against M. persicae. However, aphids fed on diets with whole cell suspensions and its cell-free supernatant of all three bacteria strains resulted in 100% mortality of adult aphids and nymphs. Specially, B. amyloliquefaciens CBMDLO3, has an effective aphicidal effect on M. persicae, used both bacterial cells and their metabolites. Moreover, heat-killed cells of B. amyloliquefaciens CBMDLO3 also had aphicidal action, although the aphid mortality was lower than on diet with living bacteria. Therefore, these results propose that B. amyloliquefaciens, could function as a novel eco-friendly biopesticide for the control of M. persicae.

Draft Genome Sequences of Lactobacillus salivarius A3iob and Lactobacillus johnsonii CRL1647, Novel Potential Probiotic Strains for Honeybees (Apis mellifera L.).

This report describes the draft genome sequences of Lactobacillus salivarius A3iob and Lactobacillus johnsonii CRL1647, probiotic strains isolated from the gut of honeybee Apis mellifera workers. The reads were generated by a whole-genome sequencing (WGS) strategy on an Illumina MiSeq sequencer and were assembled into contigs with total sizes of 2,054,490 and 2,137,413 bp for the A3iob and CRL1647 strains, respectively. The draft genome sequences of L. salivarius A3iob and L. johnsonii CRL1647 will be useful for further studies of the specific genetic features of these strains and for understanding the mechanisms of their probiotic properties.

Gram-Positive Bacteria with Probiotic Potential for the Apis mellifera L. Honey Bee: The Experience in the Northwest of Argentina.

Apis mellifera L. is one of the most important natural pollinators of significant crops and flowers around the world. It can be affected by different types of illnesses: american foulbrood, nosemosis, varroasis, viruses, among others. Such infections mainly cause a reduction in honey production and in extreme situations, the death of the colony. Argentina is the world's second largest honey exporter and the third largest honey producer, after China and Turkey. Given both the prominence of the honey bee in nature and the economic importance of apiculture in Argentina and the world, it is crucial to develop efficient and sustainable strategies to control honey bee diseases and to improve bee colony health. Gram-positive bacteria, such as lactic acid bacteria, mainly Lactobacillus, and Bacillus spp. are promising options. In the Northwest of Argentina, several Lactobacillus and Bacillus strains from the honey bee gut and honey were isolated by our research group and characterized by using in vitro tests. Two strains were selected because of their potential probiotic properties: Lactobacillus johnsonii CRL1647 and Bacillus subtilis subsp. subtilis Mori2. Under independent trials with both experimental and commercial hives, it was determined that each strain was able to elicit probiotic effects on bee colonies reared in the northwestern region of Argentina. One result was the increase in egg-laying by the queen which therefore produced an increase in bee number and, consequently, a higher honey yield. Moreover, the beneficial bacteria reduced the incidence of two important bee diseases: nosemosis and varroosis. These results are promising and extend the horizon of probiotic bacteria to the insect world, serving beekeepers worldwide as a natural tool that they can administer as is, or combine with other disease-controlling methods.