Imagine a world where we could fight back against the deadly diseases carried by mosquitoes without poisoning our planet or ourselves—sounds like science fiction, right? But this groundbreaking research uncovers a natural warrior from Bangladesh's mangrove forests that could change the game in mosquito control forever.
Bacillus altitudinis: An Eco-Friendly Biopesticide for Sustainable Mosquito Management, Derived from the Root-Zone Soil of Nypa fruticans in Mangrove Ecosystems
- Research Focus
- Freely Accessible (For more on open access science, check out resources like Springer Nature's guide to open research practices)
- Release Date: November 12, 2025
Published in BMC Microbiology (visit bmcmicrobiol.biomedcentral.com for the journal home), Volume 25, Article 733 (2025). Be sure to cite this work appropriately.
Summary
Introduction to the Problem
For centuries, mosquitoes have been humanity's silent foes, spreading devastating illnesses such as dengue fever, malaria, chikungunya, and filariasis—diseases that claim countless lives each year. In Bangladesh alone, the 2019 dengue outbreak hospitalized over 100,000 people, and sadly, these numbers keep climbing year after year, turning what was once a seasonal worry into a persistent crisis. To tackle this head-on in an environmentally responsible way, our team set out to discover and examine Bacillus bacteria strains from Bangladesh's mangrove forests and urban zones that could naturally curb mosquito populations.
Key Findings
Starting in the lab, we isolated 38 bacterial strains using a heat-shock technique and grouped them based on visual traits, Gram staining (which tells us if bacteria have a thick cell wall or not—Gram-positive ones appear purple under a microscope), and catalase tests (where bubbles signal the enzyme's presence, helping bacteria break down hydrogen peroxide). For deeper analysis in lab and live tests, we sequenced the 16S rRNA genes of 12 promising strains using universal primers 27F and 1492R—this genetic fingerprinting helps identify species accurately. We then evaluated these Bacillus strains for their ability to thrive in tough conditions through antimicrobial checks, tolerance to salt and varying pH levels, production of enzymes like amylase (which breaks down starches), cellulase (for digesting plant fibers), and protease (for protein breakdown), plus sensitivity to common antibiotics. The real highlight? Larvicidal tests against Aedes aegypti (the dengue culprit) and Culex quinquefasciatus (a filariasis vector) mosquitoes. From six standout Bacillus types—Bacillus tropicus, Bacillus thuringiensis, Bacillus zanthoxyli, Bacillus altitudinis, Bacillus pseudoflexus, and Fictibacillus barbaricus—we pinpointed top performers. Safety checks on Artemia salina (brine shrimp, often used as a stand-in for aquatic life) showed no harm from the four leading candidates. Diving into whole-genome sequencing with tools like K-mer finder, we confirmed Bacillus altitudinis as the superstar biopesticide.
Wrapping It Up
As far as we know, this marks the debut discovery of Bacillus altitudinis wielding mosquito-killing powers against larvae. With some scaling up, it holds huge promise as a marketable, planet-friendly tool for mosquito control—imagine deploying it in breeding sites without the backlash of chemicals.
Expert Reviews (Available at the article's peer review section in BMC Microbiology).
Deep Dive into the Challenge
Mosquitoes don't just buzz annoyingly; they ferry pathogens causing severe conditions like malaria, filariasis, Japanese encephalitis, dengue, chikungunya, and a slew of arboviruses including West Nile, Saint Louis encephalitis, and Eastern Equine encephalitis [1]. Over the last few decades, tropical hotspots worldwide, including Bangladesh, have seen a scary surge in these vector-borne threats. Why? Mosquitoes breed like wildfire and evolve quickly, dodging traditional controls with ease. Since the early 2000s, Bangladesh has battled yearly dengue epidemics, six of which spiked deaths past 3,000 [2]. Take 2019: In Dhaka, over 100,000 dengue cases overwhelmed hospitals, with 164 fatalities logged by the Directorate General of Health Services [3]. Then came the 2017 chikungunya wave from April to September, endangering over two million lives [4]. Dhaka Medical College Hospital tallied 690,399 confirmed cases and 291 probable ones [5]. It's clear: We need gentle, green defenses against these pests to foster lasting mosquito management that doesn't harm the environment.
Today's mosquito-fighting arsenal includes chemical sprays (think DDT, malathion, parathion, fenthion, chlorpyrifos), genetic tricks like the sterile insect technique (releasing non-breeding males to crash populations), bed nets, and traps. But chemicals? They're blunt instruments—zapping helpful bugs and wildlife alike [6,7,8]. Mosquitoes adapt fast, building resistance through genetic shifts. These persistent poisons build up in soil, rivers, and ocean floors [9,10]. For folks—especially sprayers or those in sprayed zones—overexposure brings respiratory woes, allergies, nerve damage, and even higher cancer odds [11,12]. Larvicides and insecticides ripple out, hurting humans and ecosystems; synthetic fallout is a growing red flag. That's why the World Health Organization (WHO) and Pan American Health Organization (PAHO) push biological controls—using natural 'agents' like parasites, competitors, or predators to dial down mosquito numbers [13]. But here's where it gets controversial: While biological options sound ideal, skeptics worry about unintended ecological disruptions if these agents spread unchecked. Could a 'safe' bacteria accidentally target more than just mosquitoes?
Bacteria shine brighter than harsh chemicals—they're gentler on non-target life and the planet [14]. They zero in on larvae with precision, sparing others; for instance, Bacillus thuringiensis israelensis deploys Cry toxins to gut larvae, while Bacillus sphaericus uses binary toxins [15,16]. This cuts collateral damage to fish or bees. Mosquitoes struggle to resist bacteria's varied attack styles. These microbes hang around in the wild, keeping larvae in check long-term. By hitting breeding spots, they slash adult numbers and disease spread [17]. Our project? Crafting a fresh bacterial weapon against larvae, unpacking its germ-fighting and mosquito-slaying traits. And this is the part most people miss: In resource-strapped areas like Bangladesh, scaling such biopesticides could empower local communities without relying on pricey imports.
How We Did It
Gathering Samples
We scooped up 17 eco-samples—soil, muck, wilted plants, and water—into sterile bags and jars from Khulna's Sundarbans mangroves (coordinates: 22°27’30.2”N 89°23’49.9”E) and Dhaka's Wari (23°45.04’N 90°22.79’E) and Nakhalpara (23°45.5’N 90°23.5’E) neighborhoods (see Table 1 for details). Back at the lab, we chilled them at 4°C to preserve freshness.
(Full table view available in the original source.)
Extracting and Growing Bacteria
For each gram of solid sample, we swirled it in 9 mL of distilled water in a sterile tube to free clinging bacteria. Water samples got 1 mL straight. We heat-shocked them at 80°C for 10 minutes in a Memmert water bath (Germany)—this weeds out non-spore-formers, favoring tough Bacillus. Cooled to room temp, we did serial dilutions (10−1 to 10−5). Sterilized Nutrient Agar (HIMEDIA, India) via Taisite autoclave (USA) was poured into 100 mm Petri dishes. We spread 100 µL of diluted sample with a sterile rod, then incubated at 35°C for 24 hours in a BINDER unit (Germany). Pure colonies? Streaked with loops onto fresh NA, subcultured for purity, grown 24 hours, and stored cold at 4°C.
Profiling Bacteria Visually and Biochemically
We noted colony shapes (rods, spheres?), colors, and sizes. Gram staining followed American Society of Microbiology steps: Purple means Gram-positive (thick peptidoglycan walls, common in Bacillus); pink, Gram-negative. Catalase? Bubbles from peroxide drop = positive, as these bacteria neutralize the toxin.
Genetic ID via 16S rRNA Sequencing
DNA came from the TIANamp kit protocol. We gauged purity and amount with an Eppendorf UV/Vis biophotometer (Germany). PCR amplified 16S rRNA using mini-PCR with primers: forward 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and reverse 1492R (5’-TACGGTTACCTTGTTACGACTT-3’) [18,19,20]. Products ran on Bluegel agarose; we eyed ~1500 bp bands against a ladder. Sanger sequencing (dideoxy method) at Genecreate Biotech (China) followed. Chromatograms via FinchTV led to NCBI BLAST for closest matches.
Checking Antimicrobial Power
Using agar well diffusion [21], we tested against standard strains: Salmonella typhi, Staphylococcus aureus (noted as 'Staphylococcus aeruginosa' in original, likely a typo for S. aureus), and Escherichia coli (ATCC). Swabbed Mueller Hinton agar (HIMEDIA, India, 25 mL/plate) six times, rotating 30° each. Punched 5 mm wells, added 100 µL overnight broth (0.1 or 0.5 OD turbidity). Controls: 5 µg rifampicin disc (positive), sterile water (negative). Incubated 37°C for 24–96 hours; measured inhibition zones.
Testing Salt and pH Resilience
Nutrient broths spiked with 1%, 3%, 5%, 7%, 9% NaCl or pH 2,4,6,8,10 (via HCl/NaOH). Inoculated, shook at 35°C/200 rpm for 24 hours. OD at 600 nm via C7100 spectrophotometer (Peak Instruments, USA) gauged growth. Controls: Plain broth (positive), uninoculated (negative).
Enzyme Production Checks
Amylase: Spot-inoculated on starch agar (HIMEDIA), incubated 30°C 24–96 hours. Flooded with iodine; halos = starch digestion [19].
Cellulase: Grew on CMC agar (26 g/L CMC, 15 g/L agar), 37°C for 5 days. Congo red (1% w/v) for 15 min, then 1 M NaCl; halo-colony difference in mm shows cellulose breakdown [22].
Protease: Skim milk agar (2% milk, 1% glucose, 1.8% agar, pH 9), 30°C 24–96 hours. Halos indicate proteolysis, measured like cellulase [23].
Antibiotic Sensitivity
Kirby-Bauer method [24]: Streaked 0.1 OD swabs on MH agar six times (30° rotations), dried 5–10 min. Placed discs (forceps): ciprofloxacin (5 µg), tetracycline (30 µg), vancomycin (30 µg), ampicillin (25 µg), chloramphenicol (30 µg), rifampicin (5 µg). Incubated 35°C/24 h. Zones interpreted per EUCAST: susceptible, intermediate, resistant.
Larvicidal Assays on Mosquitoes
Breeding buckets in bushes yielded Aedes and Culex larvae after 5 days [25,26]. 10 larvae per dish with 15 mL habitat water; added 5 mL bacterial culture (~1×10^8 CFU/mL from overnight NB shake at 37°C). Control: Media + water only. Monitored 48 hours, data every 24 h, analyzed in Excel.
Safety Test on Artemia salina
Hatched brine shrimp eggs in aerated saline. 10 nauplii in 100 mL saline per beaker; added 10 mL bacteria. Control: Media only. Checked lethality at 24 h intervals [27,28].
Full Genome Sequencing and Breakdown
Top isolate's DNA (TIANamp kit) quality-checked (Eppendorf at 260/280 nm). Illumina NovaSeq PE150 (Novogene, China) for paired-end 150 bp reads. FastQC (Galaxy 0.74) for quality, Trim Galore (0.6.7) for cleanup. SPAdes (Galaxy 3.15.5) de novo assembly, QUAST (5.2.0) for metrics. BV-BRC for circular genome viz.
Species Confirmation
KmerFinder 3.2 (CGE) on scaffolds: Bacterial DB, FASTA upload. Picked highest score/lowest E-value match.
Phylogenetic Tree (Codon-Based)
PATRIC's codon tree used PGFams, Mash/MinHash [29] for relatives, MUSCLE alignment [30].
Data Crunching
Excel 2019 for most; triplicates except WGS. Differential stats clarified in methods.
What We Found
From Sundarbans: 12 samples (soil, water, plants); Dhaka: 5. Yielded 38 unique isolates (Table 1), varying in colony looks (Fig. 1a).
Bacterial Traits: (a) Round white colonies, (b) Gram-positive rods, (c) Bubbling catalase positives.
(Full image in source.)
Of 38, 22 Gram-positive (selected, Fig. 1b); 16 catalase-positive (Fig. 1c). 16 dual-positives: 1T3dot, 5WR, etc. DNA from these; PCR failed for four low-DNA ones.
Gel runs showed ~1500 bp for 12 (Fig. 2a,b). Sequencing/BLAST (NCBI, April 17, 2024) matched six to Bacillus (tropicus, thuringiensis, etc.), others to Fictibacillus, Metabacillus, Halobacillus; one to Rossellomorea—hinting at diverse genetics needing more study.
Gel/PCR and Sequence Chromatogram: (a-b) Bands for isolates, (c) Clean reads.
(Full image.)
Targeted six Bacillus: 10RW, etc. Tested antimicrobial, tolerances, enzymes, antibiotics, larvicidal, lethality.
Antimicrobials: 6RW (tropicus) and 18RW (altitudinis) inhibited after 24/96 h; others nil. 6RW hit S. typhi/E. coli; 18RW S. aureus/S. typhi (Fig. 3a,b)—noted delayed 18RW effect on S. typhi, suggesting slow metabolite release.
(a) Assay plates, (b) Zone graphs.
(Full image.)
Salt: Most faltered high; 6RW topped (Fig. 4a). pH: Best 6–8, poor extremes (Fig. 4b).
(a) Salt growth curves, (b) pH optima.
(Full image.)
Enzymes: Amylase in 6RW/10RW (Fig. 5a, carbon users). Protease all six (Fig. 5b, nitrogen). Cellulase only 10RW (Fig. 5c). 10RW multi-enzyme champ (Fig. 6), aiding survival.
Enzyme Tests: (a) Amylase halos, (b) Protease, (c) Cellulase.
(Full image.)
Percent Activities Chart.
(Full image.)
Antibiotics: Cipro, tetra, vanco, chloram effective; amp/rifamp resistant in some (Fig. 7a,b). B. tropicus doubly resistant—worrisome for gene transfer?
(a) Disc plates, (b) Susceptibility table.
(Full image.)
Larvicidal: No deaths from 6RW/10RW/20W2L. 22RW: 20% at 48 h. 11RWL: 30%. 18RW: 77% (7.67±0.57, both species, Fig. 8a, Table 2). Controls: 0%. No species difference.
(Full table.)
(a) Dead larvae pics, (b) Alive Artemia.
(Full image.)
Lethality: 11RWL/22RW/18RW spared Artemia (Fig. 8b)—18RW safe for aquatics.
WGS: 9.7M trimmed reads; assembly: 32 contigs, 3.87 Mb, 41.07% GC, L50=3, no plasmids (Table 3). 100% complete (RAST).
(Full table.)
BV-BRC annotation: Close to B. altitudinis HQ-51-Ba. 4137 CDS, 3 rRNA, 68 tRNA, 5 CRISPR. Circular map: Contigs, strands, RNAs, resistance/virulence, GC/skew (Fig. 9a,b).
(a) Legend, (b) Genome circle, (c) Tree.
(Full image.)
Tree: B. altitudinis near B. pumilus NJ-M2, etc.; farthest B. psychrosaccharolyticus (distance 0.1, Fig. 9c).
Insights and Reflections
Bacterial biopesticides outshine chemicals in selectivity, low toxicity, and human safety. Prior work isolated B. amyloliquefaciens from Indian mangroves with strong larvicidal punch [31,32]—mangroves teem with resilient microbes, perfect for novel strains. Heat-shock favors spores, yielding 77 Bacillus in past studies [33].
Our 6RW/18RW antimicrobials echoed reports on Bacillus vs. Yersinia, etc. [34]. 18RW's delay? Time for metabolites.
Salt: 6RW thrived to 9%—mangrove adaptation. Past: B. aerius to 6% [35]; others low [36]. pH: Basic tolerance common.
Enzymes: Protease widespread; B. zanthoxyli triple-threat like 35% in marine study [38]. Industrial potential high, despite salt limits [36].
Antibiotics: Broad susceptibility, but resistances flag horizontal transfer risks—controversial, as beneficial bugs might fuel superbugs. Do the pros outweigh this?
WGS confirmed B. altitudinis, known for plant biocontrol [40-42]. Tools like SPAdes/BV-BRC standard [43-49].
Mosquitocidal precedents: B. amyloliquefaciens [31], B. licheniformis [50], B. sphaericus [51,52]. Our 18RW's 77% kill, zero aquatic harm? Game-changer.
Final Thoughts
Standout: 18RW (B. altitudinis) nailed 77% larval mortality, plus anti-pathogen action, antibiotic sensitivity, Artemia safety. Ideal for Aedes/Culex and S. aureus/typhi. Eco-safe first-report mosquitocide.
Drawbacks
Larvae-only; lab-scale, local larvae. Field trials needed for commercial rollout—missed here.
Data Access
PRJNA1207607 at NCBI: https://submit.ncbi.nlm.nih.gov/subs/wgs/SUB14994820/overview.
References
[Keep original list, but in rewrite, integrate as [1], etc., for brevity. Full citations in source.]
Gratitude
Thanks to BCSIR for funding/tech aid.
Support
BCSIR R&D 2023-24.
Team
Md. Saddam Hossain¹ (lead design/editor), Motahara Farhan Anjum¹ (co-first, experiments/draft), Md. Asik Rabbani² (assists/writing), Md. Rakibul Hasan³ (larval tests), Md. Mehadi Hasan Sohag² (edits/guidance), Nishat Tasnim³ (experiments/edits), Debabrata Karmakar³ (chems/edits), Sharmin Akter⁴ (edits/collection), Md. Masudur Rahman⁵ (samples/edits), Md. Rezaul Karim³ (guidance).
Affiliations: ¹Chattogram Veterinary University; ²Jagannath University; ³BCSIR; ⁴Jashore University; ⁵Bangladesh Fisheries.
Contact: Md. Saddam Hossain.
Ethics: No human/animal tests; BCSIR approved.
No conflicts.
Publisher Note: Neutral on claims.
Open Access: CC BY-NC-ND 4.0—share non-commercially, credit source.
About: Cite as Hossain et al., BMC Microbiol 25:733 (2025). DOI: 10.1186/s12866-025-04168-0.
Received March 19, 2025; Accepted June 24, 2025; Published Nov 12, 2025.
Keywords: [Original list].
What do you think—could Bacillus altitudinis revolutionize global mosquito control, or are there hidden risks we haven't considered? Share your take in the comments: Agree it's a breakthrough, or worried about resistance spreading? Let's discuss!
