Assessment of the Synergic Effect between Lysinibacillus sphaericus S-Layer Protein and Glyphosate in the Lethality of the Invasive Arboviral Vector Aedes albopictus.

Glyphosate and glyphosate-based herbicides are among the most used chemicals in plant pest control. Both glyphosate and its main by-product Aminomethylphosphonic Acid (AMPA) are highly environmentally persistent and, through several processes (including surface runoff and bioaccumulation), affect species beyond their intended targets, especially in aquatic ecosystems. Aedes albopictus is a novel invasive arboviral vector in Colombia and has spread to much of the national territory in recent years. Strains of the bacterium Lysinibacillus sphaericus have shown the ability to degrade glyphosate into environmentally inert compounds, in addition to having great larvicidal efficiency in different mosquito species through the production of several proteins, including the surface layer (S-Layer) protein. The S-Layer is a bacterial structure consisting of glycoprotein monomers, and its functions are thought to include bacterial interactions, protection from the outside medium and biological control. The study assessed the entomopathogenic activity of L. sphaericus S-Layer protein on Ae. albopictus larvae, and the effects that glyphosate and its by-products have in this process. To that end, bioassays were performed to compare the larval mortality between different treatments with and without S-Layer, glyphosate, and glyphosate derivates. Comparisons were made through Analysis of variance (ANOVA) and Tukey's Honestly Significant Difference (HSD) analyses. Significant differences were found in larval mortality in the treatments, and larval mortality was greater when the S-Layer protein was present, though glyphosate field-doses (1.69 g/L) alone had a notable toxicity as well. An apparent synergic effect on the mortality of larval Ae. albopictus when exposed to mixtures containing 1500 ppm of the S-Layer protein, glyphosate, and/or glyphosate derivates was found. Further studies are needed for the in-depth understanding of this mechanism and its consequences on aquatic ecosystems.

Effective Gold Biosorption by Electrospun and Electrosprayed Bio-composites with Immobilized Lysinibacillus sphaericus CBAM5.

Electro-hydrodynamic processing, comprising electrospinning and electrospraying techniques, is a novel technology used in the production of nano- and sub-micro-scale materials with specific properties suitable for environmental remediation processes. Polycaprolactone (PCL) micro-fibrous mats and alginate microcapsules were produced using electrospinning and electrospraying techniques respectively, and Lysinibacillus sphaericus CBAM5, a bacterium capable of metal removal by adsorption and accumulation inside the cell, was immobilized in these matrices. The polymeric structure was able to protect and maintain cell viability and the bio-composite materials were used to capture gold from synthetic water samples. The micro-fibrous membranes with immobilized bacteria were able to remove 93% of the gold after 120 h of inclusion in the aqueous medium. Using a filtration system, an efficiency of 64% was obtained for the removal of the precious metal after 10 cycles of filtration (2 h of exposure to the gold solution). In contrast, the microencapsulated L. sphaericus CBAM5 captured 64% of the gold after 4 h of the assay. Thus, both micro-structured matrices were suitable for the immobilization and protection of L. sphaericus CBAM5 and they showed high efficiencies of gold biosorption. Hence, these bio-composite materials could be used to concentrate gold from industrial wastewaters.

Synergistic effect of Lysinibacillus sphaericus and glyphosate on temephos-resistant larvae of Aedes aegypti.

BACKGROUND: Glyphosate-based herbicides are one of the most commonly used compounds to control perennial weeds around the world. This compound is very persistent in the environment and tends to filter into aquatic ecosystems, affecting non-target species such as mosquito larvae. Aedes aegypti mosquitoes are vectors of multiple arboviruses such as dengue and Zika. Glyphosate can be degraded into non-harmful environmental compounds by Lysinibacillus sphaericus, a spore forming bacterium which can also kill Ae. aegypti larvae. In this study, we assessed the effect of glyphosate concentrations, typically used in Colombia, on the entomopathogenic activity of L. sphaericus against Ae. aegypti larvae. METHODS: Bioassays and toxicity curves were performed to compare the larval mortality between different treatments with and without bacteria and glyphosate (Roundup 747®). Larvae were exposed to both bacteria and glyphosate by adding the compound on chloride-free water. Comparisons were made using both probit regression and ANOVA analysis. RESULTS: ANOVA showed a significant difference in larval mortality when adding glyphosate and L. sphaericus at the same time. Thus, a positive synergic effect on larval mortality was found when L. sphaericus and glyphosate were mixed. According to probit analysis, median lethal dose (LD50) for bacterial mixture was of 106.23 UFC/ml and for glyphosate was 2.34 g/l. CONCLUSIONS: A positive synergic effect on the mortality of larval Ae. aegypti when exposed to L. sphaericus mixture and glyphosate was found. Molecular studies focusing on the toxin production of L. sphaericus are required to understand more about this synergistic effect.

The Golden Activity of Lysinibacillus sphaericus: New Insights on Gold Accumulation and Possible Nanoparticles Biosynthesis.

Power struggles surrounding the increasing economic development of gold mining give rise to severe environmental and social problems. Two new strains of Lysinibacillus sphaericus were isolated from an area of active alluvial gold mining exploitation at El Bagre, Antioquia. The absorption capacity of these strains and some of the L. sphaericus Microbiological Research Center (CIMIC) collection (CBAM5, OT4b.31, III(3)7) were evaluated by spectrophotometry according to a calibration gold curve of HAuCl4- with concentrations between 0 µg/mL and 100 µg/mL. Bioassays with living biomass were carried out with an initial gold concentration of 60 µg/mL. Their sorption capacity was evident, reaching percentages of gold removal between 25% and 85% in the first 2 h and 75% to 95% after 48 h. Biosynthesis of possible gold nanoparticles (AuNPs) in assays with living biomass was also observed. Metal sorption was evaluated using scanning electron microscopy and energy-dispersive X-ray spectroscopy (EDS) analysis. The sorption and fabrication capacity exhibited by the evaluated strains of L. sphaericus converts this microorganism into a potential alternative for biomining processes, especially those related to gold extraction.

Molecular assessment of glyphosate-degradation pathway via sarcosine intermediate in Lysinibacillus sphaericus.

The widespread use of glyphosate has permeated not only small- and large-scale agriculture, but also the fight against drug trafficking and illicit crops. Health, alimentary security, and the rights of peasant and indigenous communities have been compromised in countries with intensive use of glyphosate-based herbicides. In 2015, the International Agency for Research on Cancer classified this substance as probably carcinogenic to humans, leading to the suspension of aerial glyphosate spraying the same year in countries like Colombia, where glyphosate has been extensively used in illicit crop eradication. Notwithstanding, according to a study of the U.S. Geological Survey, traces of glyphosate and its main degradation product, AMPA, remain in soil year after year. This underscores the urgency and importance of assessing new technologies to degrade glyphosate present in soils and waterbodies without leaving persistent byproducts. The aim of this study was to evaluate Lysinibacillus sphaericus' glyphosate uptake as a carbon and phosphorous source by a sarcosine-mediated metabolic pathway that releases glycine as final degradation product. To accomplish this, molecular and analytic evidence were collected in vitro from sarcosine oxidase activity, a key enzyme of a degradation pathway which releases byproducts that are easy to incorporate into natural biosynthesis routes.

Asymmetrical Competition between Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae) Coexisting in Breeding Sites.

Aedes aegypti and Culex quinquefasciatus are mosquito vectors for several tropical diseases that represent a current public health problem. The ecological requirements for each species are different, however, both species show high biological adaptability, which promotes their coexistence in the same breeding sites. The purpose of this study was to assess the effect of larval association between Ae. aegypti and Cx. quinquefasciatus under different laboratory conditions of food supply and temperature, and under field simulated conditions like peridomestic containers. Our findings showed that under field simulated conditions there was no asymmetrical competition in mixed cultures with the different Cx. quinquefasciatus/Ae. aegypti ratios tested. However, under laboratory conditions in which different doses of food supply were evaluated, it was observed that competition between the two species takes place. Larval coexistence under food scarcity conditions (0.95 mg/larva) showed that Ae. aegypti had a greater adult emergence than Cx. quinquefasciatus and was capable of depriving Cx. quinquefasciatus of the food needed to complete metamorphosis. In an intermediate dose of food (1.9 mg/larva), the dry weight of Cx. quinquefasciatus adults decreased, and their larval development time increased when Cx. quinquefasciatus/Ae. aegypti ratio was low. Also, a temperature effect was assessed demonstrating that Cx. quinquefasciatus was more vulnerable to changes in temperature. We suggest that Ae. aegypti is more successful in exploiting microhabitats when food is scarce, due to its scrape active feeding habitats and fast larval development times. Therefore, in conditions of food paucity both species will compete, and Ae. aegypti larvae will prevail.

Contribution of Lysinibacillus sphaericus hemolysin and chitin-binding protein in entomopathogenic activity against insecticide resistant Aedes aegypti.

Given its toxicity against culicid larvae, Lysinibacillus sphaericus is used for the biological control of mosquitoes such as Culex sp. and Anopheles sp. The toxicity factors currently reported for L. sphaericus include the Binary toxin, Mtx toxins, and the S-layer. However, Aedes aegypti is refractory to the Binary toxin, the most toxic larvicidal protein of L. sphaericus. Until now, there are no evidences of the hemolytic and chitinolytic capacity of L. sphaericus. Herein, the expression of the hemolysin D (hlyD) and the chitin-binding protein genes of L. sphaericus III(3)7, OT4b.25, and 2362 was quantified. Gene expression was assessed 24 and 48 h after field-collected and Rockefeller A. aegypti larvae were fed with the bacteria. The hlyD gene showed the highest expression at 24 h whilst the expression of the chitin-binding protein gene increases at 48 h. The highest hlyD expression was seen in the III(3)7 strain and the highest chitin-binding protein gene expression was in the 2362 strain. The consortium of L. sphaericus III(3)7 and 2362 showed the highest expression of both genes being with field-collected and Rockefeller larvae. The results suggest that hemolysin D and the chitin-binding protein can be two novel toxic elements involved in the entomopathogenic activity of L. sphaericus. These proteins, along with the other L. sphaericus toxins, make this bacterium a suitable alternative to replace the chemical insecticides used for the control of A. aegypti populations.

A genome-scale metabolic reconstruction of Lysinibacillus sphaericus unveils unexploited biotechnological potentials.

The toxic lineage (TL) of Lysinibacillus sphaericus has been extensively studied because of its potential biotechnological applications in biocontrol of mosquitoes and bioremediation of toxic metals. We previously proposed that L. sphaericus TL should be considered as a novel species based on a comparative genomic analysis. In the current work, we constructed the first manually curated metabolic reconstruction for this species on the basis of the available genomes. We elucidated the central metabolism of the proposed species and, beyond confirming the reported experimental evidence with genomic a support, we found insights to propose novel applications and traits to be considered in further studies. The strains belonging to this lineage exhibit a broad repertory of genes encoding insecticidal factors, some of them remain uncharacterized. These strains exhibit other unexploited biotechnological important traits, such as lactonases (quorum quenching), toxic metal resistance, and potential for aromatic compound degradation. In summary, this study provides a guideline for further research aimed to implement this organism in biocontrol and bioremediation. Similarly, we highlighted the unanswered questions to be responded in order to gain a deeper understanding of the L. sphaericus TL biology.

Efficacy of the vegetative cells of Lysinibacillus sphaericus for biological control of insecticide-resistant Aedes aegypti.

BACKGROUND: The control of Aedes aegypti is usually based on chemical insecticides, but the overuse of these compounds has led to increased resistance. The binary toxin produced by Lysinibacillus sphaericus in the final stages of sporulation is used for mosquito control due to its specificity against the culicid larvae; however, it has been proved that Ae. aegypti is refractory for this toxin. Currently, there is no evidence of the use of L. sphaericus vegetative cells for mosquito biocontrol. Therefore, in this study, the vegetative cells of three L. sphaericus strains were assessed against a field-collected Ae. aegypti, resistant to temephos, and the reference Rockefeller strain. RESULTS: Vegetative cells of L. sphaericus 2362, III(3)7 and OT4b.25 produced between 90% and 100% of larvae mortality in the reference Rockefeller strain. Effective concentrations of each L. sphaericus strain for the four larval stages ranged from 1.4 to 2 × 107 CFU/ml. Likewise, a consortium of L. sphaericus assessed against a field-collected Ae. aegypti resistant to temephos and the Rockefeller strain caused 90% of larvae mortality. Concentrations of L. sphaericus consortium that resulted in larvae mortality of field-collected and Rockefeller Ae. aegypti ranged from 1.7 to 2.5 × 107 CFU/ml. The vegetative cells of L. sphaericus have no effect on the Ae. aegypti eggs and pupae. CONCLUSIONS: The vegetative cells of L. sphaericus are effective against Ae. aegypti larvae, meaning that it could be used in the biological control of these mosquito species. Since the L. sphaericus consortium was effective against temephos-resistant Ae. aegypti, vegetative cells could be an alternative to overcome insecticide-resistant populations. Further studies, should be conducted to reveal the mode of action and the toxic principle of L. sphaericus vegetative cells.

Synergistic Activity Between S-Layer Protein and Spore-Crystal Preparations from Lysinibacillus sphaericus Against Culex quinquefasciatus Larvae.

Lysinibacillus sphaericus is used for the biological control of mosquitoes. The main toxicity mechanism of pathogenic strains is a binary toxin produced during sporulation. S-layer is a proteinaceous structure on the surface of bacteria; its functions have been involved in the interaction between bacterial cells and the environment, for example, as protective coats, surface recognition, and biological control. In L. sphaericus, S-layer protein (SlpC) is expressed in vegetative cells, and is also found in spore-crystal preparations; it has larvicidal activity in Culex spp. In this study, partial and completed sporulated culture toxicities were compared; also, S-layer protein and spore-crystal proteins were tested against Culex quinquefasciatus larvae for possible interactions. Larvicidal activity obtained with a combination of SlpC and spore-crystal proteins from strain III(3)7 showed no significant interaction, whereas, combinations of both preparations from strain 2362 showed synergistic effect. The highest synergistic activity observed was between spore protein complex from strain 2362 and SlpC from III(3)7. S-layer protein could be considered a good alternative in formulation improvement, for biological control of mosquitoes.

Evidence-based validation of quorum quenching from Lysinibacillus sphaericus and Geobacillus sp. in bioremediation of oil sludge.

Many studies on quorum quenching focus on the discovery and characterization of novel acyl-homoserine lactonases (AHL-lactonases) because these enzymes could be used in the control of diseases caused by Gram-negative bacteria. The effects of quorum quenching are also remarkable in the performance of bacterial consortia in applications such as bioremediation. In the current work, we demonstrated the presence of a potential novel AHL-lactonase-encoding locus (Bsph_3377) from Lysinibacillus sphaericus and Geobacillus sp. The deduced amino acid sequences for this enzyme possess the characteristic domains and motifs involved in Zn-binding from AHL lactonases and were grouped into 1 clade within the phylogeny of the lactonases from firmicutes, showing 70% of identity with the lactonase AhlS from Solibacillus silvestris. We demonstrated the locus transcription by RT-qPCR and its relationship with the suppression of the pathogenicity of Pectobacterium carotovorum. Additionally, we analyzed the interaction of these bacilli with a commercial consortium in the bioremediation of a hydrocarbon-contaminated soil, showing inhibitory effects on its establishment. These results represent a new contribution in the understanding of the potential biotechnological applications of L. sphaericus and Geobacillus sp. as well as in the research on antibacterial techniques based on quorum-sensing disruption.

Comparative genomics reveals Lysinibacillus sphaericus group comprises a novel species.

BACKGROUND: Early in the 1990s, it was recognized that Lysinibacillus sphaericus, one of the most popular and effective entomopathogenic bacteria, was a highly heterogeneous group. Many authors have even proposed it comprises more than one species, but the lack of phenotypic traits that guarantee an accurate differentiation has not allowed this issue to be clarified. Now that genomic technologies are rapidly advancing, it is possible to address the problem from a whole genome perspective, getting insights into the phylogeny, evolutive history and biology itself. RESULTS: The genome of the Colombian strain L. sphaericus OT4b.49 was sequenced, assembled and annotated, obtaining 3 chromosomal contigs and no evidence of plasmids. Using these sequences and the 13 other L. sphaericus genomes available on the NCBI database, we carried out comparative genomic analyses that included whole genome alignments, searching for mobile elements, phylogenomic metrics (TETRA, ANI and in-silico DDH) and pan-genome assessments. The results support the hypothesis about this species as a very heterogeneous group. The entomopathogenic lineage is actually a single and independent species with 3728 core genes and 2153 accessory genes, whereas each non-toxic strain seems to be a separate species, though without a clear circumscription. Toxin-encoding genes, binA, B and mtx1, 2, 3 could be acquired via horizontal gene transfer in a single evolutionary event. The non-toxic strain OT4b.31 is the most related with the type strain KCTC 3346. CONCLUSIONS: The current L. sphaericus is actually a sensu lato due to a sub-estimation of diversity accrued using traditional non-genomics based classification strategies. The toxic lineage is the most studied with regards to its larvicidal activity, which is a greatly conserved trait among these strains and thus, their differentiating feature. Further studies are needed in order to establish a univocal classification of the non-toxic strains that, according to our results, seem to be a paraphyletic group.

Toxic metals and associated sporulated bacteria on Andean hummingbird feathers.

Human activities in the Sabana de Bogotá, Colombia, release toxic metals such as lead (Pb) and chromium (Cr) into the environment polluting the air, water, and soil. Because birds are in contact with these pollutants and their sources, they may serve as bioindicator organisms. We evaluated the use of hummingbird feathers obtained from individuals captured in three sites of the Sabana de Bogotá as bioindicators of toxic metal pollution using spectrophotometric and spectroscopic methods based on single-feather samples. We also characterized the bacterial microbiota associated with hummingbird feathers by molecular identification using the 16S rRNA with a special focus on sporulated bacteria. Finally, we described the interactions which naturally occur among the feathers, their associated bacteria, and pollutants. We found differences in Pb and Cr concentrations between sampling sites, which ranged from 2.11 to 4.69 ppm and 0.38 to 3.00 ppm, respectively. This may reflect the impact of the activities held in those sites which release pollutants to the environment. Bacterial assemblages mainly consisted of sporulated bacilli in the Bacillaceae family (65.7 % of the identified morphotypes). We conclude that the feathers of wild tropical birds, including hummingbirds, can be used as lead and chromium bioindicators and that bacteria growing on feathers may in fact interact with these two toxic metals.

Complete genome sequencing and comparative genomic analysis of functionally diverse Lysinibacillus sphaericus III(3)7.

Lysinibacillus sphaericus III(3)7 is a native Colombian strain, the first one isolated from soil samples. This strain has shown high levels of pathogenic activity against Culex quinquefaciatus larvae in laboratory assays compared to other members of the same species. Using Pacific Biosciences sequencing technology we sequenced, annotated (de novo) and described the genome of strain III(3)7, achieving a complete genome sequence status. We then performed a comparative analysis between the newly sequenced genome and the ones previously reported for Colombian isolates L. sphaericus OT4b.31, CBAM5 and OT4b.25, with the inclusion of L. sphaericus C3-41 that has been used as a reference genome for most of previous genome sequencing projects. We concluded that L. sphaericus III(3)7 is highly similar with strain OT4b.25 and shares high levels of synteny with isolates CBAM5 and C3-41.

Complete Genome Sequence of Lysinibacillus sphaericus WHO Reference Strain 2362.

Lysinibacillus sphaericus is a species that contains strains widely used in the biological control of mosquitoes. Here, we present the complete 4.67-Mb genome of the WHO entomopathogenic reference strain L. sphaericus 2362, which is probably one of the most commercialized and studied strains. Genes coding for mosquitocidal toxin proteins were detected.

Complete Genome Sequence of the Larvicidal Bacterium Lysinibacillus sphaericus Strain OT4b.25.

Lysinibacillus sphaericus OT4b.25 is a native Colombian strain isolated from coleopteran larvae in an oak forest near Bogotá D.C.; this strain has shown high levels of pathogenic activity against Culex quinquefasciatus larvae in laboratory assays compared to that of other members of the same species. Using Pacific Biosciences sequencing technology, we propose a chromosomal contig of 4,665,775 bp that, according to comparative analysis, is highly similar to that of reference strain L. sphaericus C3-41.

Adsorption of Toxic Metals and Control of Mosquitos-borne Disease by Lysinibacillus sphaericus: Dual Benefits for Health and Environment / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Assessment of the bacterium L. sphaericus as a dual-action candidate for biological control of mosquito-borne diseases and bioremediation of toxic metals.</p><p><b>METHODS</b>Larvae of the mosquito, C. quinquefasciatus, were first evaluated for metal tolerance and then exposed to 5 ppm cadmium, chromium, arsenic, and lead in assays together with seven strains of L. sphaericus. A probit regression analysis was used to estimate the LC(50) of Cd, Cr, As, and Pb to C. quinquefasciatus. An analysis of covariance and multifactorial ANOVA examined the metal biosorption and larvicidal properties of the seven strains of L. sphaericus.</p><p><b>RESULTS</b>We found that L. sphaericus adsorbed the toxic metal ions and was toxic against mosquito larvae. The L. sphaericus strain III(3)7 resulted in a larvae mortality of over 80% for all the tested metals. This strain also exhibited the capacity to adsorb 76% of arsenic, 32% of lead, 25% of chromium, and 7% of cadmium.</p><p><b>CONCLUSION</b>This study found combined metal adsorption and larval toxicity associated with three strains of L. sphaericus [III(3)7, OT4b.31, and CBAM5]. This suggests that a combination of these strains shows strong dual potential for biological control of mosquitos in heavy metal-contaminated areas and remediate the heavy metal contamination as well.</p>

Genome sequence and description of the mosquitocidal and heavy metal tolerant strain Lysinibacillus sphaericus CBAM5.

Lysinibacillus sphaericus CBAM5, was isolated from subsurface soil of oil well explorations in the Easter Planes of Colombia. This strain has potential in bioremediation of heavy-metal polluted environments and biological control of Culex quinquefasciatus. According to the phylogenetic analysis of 16S rRNA gene sequences, the strain CBAM5 was assigned to the Lysinibacillus sphaericus taxonomic group 1 that comprises mosquito pathogenic strains. After a combination assembly-integration, alignment and gap-filling steps, we propose a 4,610,292 bp chromosomal scaffold. The whole genome (consisting of 5,146,656 bp long, 60 contigs and 5,209 predicted-coding sequences) revealed strong functional and syntenial similarities to the L. sphaericus C3-41 genome. Mosquitocidal (Mtx), binary (Bin) toxins, cereolysin O, and heavy metal resistance clusters from nik, ars, czc, mnt, ter, cop, cad, and znu operons were identified.

Metal tolerance and larvicidal activity of Lysinibacillus sphaericus.

Lysinibacillus sphaericus is a spore-forming bacterium used in the biological control of mosquitoes and in bioremediation. Mosquito larvae exposed to heavy metals are tolerant to concentrations above the permissible limit for industrial residual waters. In this work, we characterize 51 L. sphaericus strains for metal tolerance and larvicidal activity against Culex quinquefasciatus. Lysinibacillus sphaericus OT4b.2, OT4b.20, OT4b.25, OT4b.26 and OT4b.58 were as toxic as the spores of the reference strain 2362 against C. quinquefasciatus larvae. 19 Mosquito-pathogenic L. sphaericus strains and 6 non-pathogenic strains were able to grow in arsenate, hexavalent chromium and/or lead. 16S rRNA gene sequences and phylogenetic analyses clustered 84 % of the metal-tolerant strains in L. sphaericus group 1, which encompasses the mosquitocidal strains. The larvicidal activity of vegetative and sporulated cells and its high tolerance to arsenate, hexavalent chromium and lead indicate that L. sphaericus OT4b.26 is a strong candidate for further studies examining its potential for biological control of mosquitoes in waters contaminated with metals.