Taxonomic Diversity and Antimicrobial Potential of Thermophilic Bacteria from Two Extreme Algerian Hot Springs

Abstract

This study investigated thermophilic bacterial communities from two Algerian hot springs: Hammam Debagh (94-98 °C), recognized as the second hottest spring in the world, and Hammam Bouhadjar (61-72 °C), one of the hottest in northwest Algeria. Thirty isolates were obtained, able to grow between 45 °C and 80 °C, tolerating pH 5.0-12.0 and NaCl concentrations up to 3%. Colonies displayed diverse morphologies, from circular and smooth to star-shaped and Saturn-like forms. All isolates were characterized as Gram-positive, catalase-positive rods or filamentous bacteria. Identification by MALDI-TOF, rep-PCR and 16S rRNA sequencing classified them mainly within Bacillus, Brevibacillus, Aneurinibacillus, Geobacillus, and Aeribacillus, with Geobacillus predominating. Rep-PCR provided higher resolution, revealing intra-species diversity overlooked by MALDI-TOF MS and 16S rRNA. A subset of six isolates, mainly Geobacillus spp., was selected based on phenotypic and genotypic diversity and tested for antimicrobial activity against thermophilic target isolates from the same hot spring environments. Strong inhibition zones (~24 mm) were observed, with Geobacillus thermoleovorans B8 displaying the highest activity. Optimization on Modified Nutrient Agar medium with Gelrite enhanced antimicrobial production and inhibition clarity. These findings highlight the ecological and biotechnological significance of thermophilic bacteria from Algerian geothermal ecosystems. While this study focused on microbial interactions within thermophilic communities, the promising inhibitory profiles reported here provide a foundation for future research targeting foodborne and antibiotic-resistant pathogens, as part of broader efforts in biopreservation and sustainable antimicrobial development.

Keywords: 16S rRNA; MALDI-TOF MS; antimicrobial peptides (AMPs); biotechnological applications; extreme environments; rep-PCR fingerprinting; thermophiles.

Figure 1

Macroscopic appearance and morphological diversity of thermophilic bacterial isolates (G and B isolates) grown on Nutrient Agar, originating from two Algerian hot springs: Hammam Debagh (G) and Hammam Bouhadjar (B).

Figure 2

Comparative analysis of similarity index distributions and scores among thermophilic isolates assessed by (GTG)₅-PCR, BOX-PCR, and ERIC-PCR techniques. Clustered multidimensional scaling (MDS) representations were generated to visualize bacterial strain similarities for each method: (GTG)₅-PCR (A), BOX-PCR (B), and ERIC-PCR (C), with numbers representing unique isolates and colors indicating genetic clusters. A box plot (D) compares the similarity scores obtained across the three techniques, highlighting differences in variability, central tendency, and discriminatory resolution among the methods.

Figure 3

UPGMA dendrogram showing the genetic relatedness of 30 thermophilic bacterial isolates from Hammam Debagh and Hammam Bouhadjar hot springs based on (GTG)₅-PCR fingerprinting profiles using (GTG)₅ primer.

Figure 4

UPGMA dendrogram showing the genetic relatedness of 30 thermophilic bacterial isolates from Hammam Debagh and Hammam Bouhadjar hot springs based on BOX-PCR fingerprinting profiles using BOXA1R primer.

Figure 5

UPGMA dendrogram showing the genetic relatedness of 30 thermophilic bacterial isolates from Hammam Debagh and Hammam Bouhadjar hot springs based on ERIC-PCR fingerprinting profiles using ERIC 1R and ERIC 2 primers.

Figure 6

Phylogenetic tree based on 16S rRNA gene sequences of 30 thermophilic isolates (regular font) and 12 reference isolate strains (in bold font, along with their NCBI accession numbers), constructed using the Maximum Likelihood method [51] with MEGA12 software (v12.0.8) [55]. Evolutionary relationships were inferred under the 2-parameter nucleotide substitution model (+Γ, 5 categories; +I, 8.96% invariant sites). Bootstrap support values (500 replicates) [52] are shown along branches. Sequences were aligned using MUSCLE [56] to ensure high accuracy.

Figure 7

Agar well diffusion assay showing inhibition zones (clear halos) around wells containing thermophilic bacterial isolates. Six isolates (B16, B30, B70, B8, G14, and G4) were tested against six target isolates (G10, B18, B9, G5, B15, and B70). Wells are labeled according to isolate numbers (16, 30, 70, 8, 14, 4) on each Petri dish, which is labeled with the corresponding target isolate. The size of the clear halo around each well reflects the antimicrobial potency of the isolate. Inhibition zone diameters were measured to compare the effectiveness of the isolates.