Coexistence of Heavy Metal Tolerance and Antibiotic Resistance in Thermophilic Bacteria Belonging to Genus Geobacillus

Abstract

Hot springs are thought to be potential repositories for opportunistic infections, such as antibiotic-resistant strains. However, there is a scarcity of information on the mechanisms of antibiotic resistance gene (ARG) uptake, occurrence, and expression in thermophilic bacteria. Furthermore, because the genesis and proliferation of ARGs in environmental microorganisms are unknown, the research on antibiotic resistance profiles and probable mechanisms in thermophilic bacteria will become increasingly important. The goals of this study are to explore bacterial diversity, antibiotic and heavy metal resistance, and the prevalence and presence of ARG and metal resistance gene (MRG) in Geobacillus species. The 16S rRNA sequencing was used to determine the culturable bacterium diversity of 124 isolates. Standard Kirby Bauer Disc Diffusion and tube dilution procedures were used to determine antibiotic sensitivity and minimum inhibitory concentration (MIC). The tube dilution method was also used to check metal tolerance. To detect ARG and heavy MRG (HMRG), whole genome sequencing studies of the type species of the genus Geobacillus and five randomly selected Geobacillus species were performed. Graph Pad Prism and XLSTAT were used to perform statistical analyses such as ANOVA, EC50 analysis, and principal component analysis (PCA). The phylum Firmicutes and the genus Geobacillus dominated the culture-dependent bacterial diversity. Surprisingly, all thermophilic isolates, i.e., Geobacillus species, were sensitive to at least 10 different antibiotics, as evidenced by the lack of ARGs in whole genome sequencing analysis of numerous Geobacillus species. However, some of these isolates were resistant to at least five different heavy metals, and whole genome sequencing revealed the presence of MRGs in these thermophilic bacteria. The thermophilic genus Geobacillus is generally antibiotic sensitive, according to this study. In contrast, heavy metal is tolerated by them. As a result, it is possible that ARGs and MRGs do not coexist in these bacteria living in hot springs.

Keywords: Geobacillus; Sikkim Himalaya; antibiotics resistance; heavy metal tolerance; thermophilic microbes.

Figure 1

(A) Phylogenetic tree of Reshi isolates: The evolutionary history was inferred using the Maximum Likelihood method. The bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary history of the taxa analyzed. The evolutionary distances were computed using the Jukes-Cantor method and are in the units of the number of base substitutions per site. Evolutionary analyses were conducted in MEGA11. Bacillus subtilis DSM 10T (NR 027552.1) was taken as an outgroup. Geobacillus isolates were colored red and Parageobacillus isolates were colored as blue. (B) Phylogenetic tree of Yumthang isolates: The evolutionary history was inferred using the Maximum Likelihood method. The bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary history of the taxa analyzed. The evolutionary distances were computed using the Jukes-Cantor method and are in the units of the number of base substitutions per site. Evolutionary analyses were conducted in MEGA11. Bacillus subtilis DSM 10T (NR 027552.1) was taken as an outgroup. Geobacillus isolates were colored red and Parageobacillus isolates were colored as blue.

Figure 2

Minimum inhibitory concentration shown by various antibiotics in μg/ml.

Figure 3

Minimum inhibitory concentration showing by isolates toward various heavy metals.

Figure 4

Principal component analysis showing the correlation of heavy metals and isolated strains.

Figure 5

Comparison of minimum inhibitory concentrations among studied isolates and two known Geobacillus species and an E. coli.