## 3 Discussion  
The emergence of the SmR phenotype in bacteria readily occurred to a large extent because of the wide commercial use of streptomycin as medical treatment to control microbial pathogens and as an animal feed additive for growth promotion. Streptomycin has improved in efficiency for over 50 years, although recently, the antibiotic has been substituted by various alternatives, such as prebiotics, probiotics, synbiotics, and antimicrobial peptides [31; 32]. In fact, the three types of streptomycin phenotypes in bacteria, namely, SmR, SmP, and SmD, are the result of substituted amino acid residues at the site of streptomycin interaction [11]. Interestingly, a well-known probiotic, _Lactobacillus_ species, has extremely high MIC levels after low-level exposure to streptomycin. The strain has an _rpsL_ gene mutation, and the accumulation of mutations through antibiotic pressure resulted in high-level resistance exceeding 131,000 ug/mL streptomycin after 25 days. This SmR strain quickly lost the SmR phenotype without antibiotic pressure but maintained resistance to 2040 ug/mL streptomycin after 35 days [33]. Similarly, _E. coli_ isolated from an environmental sea product revealed resistance to more than 1024 ug/mL streptomycin [34]. Laboratory experiments in evolution have also shown a high level of antibiotic resistance stemming from antibiotic exposure in different bacteria [3; 35; 36]. In fact, various bacteria readily gained streptomycin resistance with a wide range of MICs. Since the analysis of _strA_ (which encodes an aminoglycosoghodtransferase) point mutations in _E. coli_ in  
Figure 6: qRT-PCR for Sm-enhanced clone growth of _dgeo_2840 parent and two SmD strains (S1 and S2) with the wild type at OD\({}_{600}\) of 2.0 and 4.0. _dgeo_0646_ and _dgeo_1869_ are EF-Tu genes (_tufA_ and _tufB_, respectively), and _dgeo_1202_ is a putative _audA_ gene. The statistics were performed with a probability \(t\)-test in the Prism program with \(p<0.05\) (*), \(p<0.001\) (**), and \(p<0.0001\) (***). 1972, there has been significant research into SmR mechanisms, including the analysis of various point mutations and structural resolutions of conformational changes to ribosomal proteins in _E. coli_, _Salmonella_ Typhimurium, and _T. thermophilus_[4, 37, 38, 39]. Following the understanding of the translational machinery and ribosomal structure, as well as the development of different protein mutation assays, the mechanisms underlying resistance to aminoglycosad antibiotics have been defined for the past half-century. Two helices of 16S rRNA, h18 (525 th and 526 th nucleotides) and h27 (912 th and 913 th nucleotides) of the central pseudoknot area of 16S rRNA, interact with two loop regions, namely, K43K44P45 and K88 of ribosomal protein S12, as shown in Figure S2 [40]. A loop of the S12 protein interacts with the tRNA anticodon stem-loop structure at the 'A' site and stabilizes the bases of 16S rRNA that are involved in the recognition of codon-anticodon base pairing [41]. Streptomycin binds between the 16S rRNA and S12 interaction area, resulting in translational misreading [37]. A deficiency in N-7 methylation of G527 of 16S rRNA by the methyltransferase RsmG produces low-level streptomycin resistance. Point and frameshift mutations in the 252 loop region of _rsmG_ inactivate 16S rRNA methyltransferase activity, resulting in a moderate SmR phenotype in the range of several 100 \(\upmu\)g/mL. In particular, the K43, K44, and K88 lysine residues of ribosomal protein S12 (RpsL) are directly involved in this interaction with streptomycin [4]. Further, the tertiary interaction between the 16S rRNA helices h44 and h45 is destabilized by streptomycin interaction [5]. Thus, if this region is mutated, the mutant strains reveal a higher MIC level of resistance exceeding 32,000 \(\upmu\)g/mL [13]. Additionally, _mthA_ is involved in the low-level SmR phenotype [42].