Tetracycline is an organic compound with the molecular formula C22H24N2O8. It and its salts are yellow or light yellow crystals. They are extremely stable in a dry state. Except for chloramphenicol, the aqueous solutions of other tetracyclines are quite stable. Tetracyclines are soluble in dilute acids and alkalis, slightly soluble in water and lower alcohols, but insoluble in ether and petroleum ether.
A new tetracycline (TC)-degrading bacterium, Providencia stuartii TX2, was isolated from the intestine of black soldier fly larvae (BSFL), achieving efficient removal of TC in different environments, revealing the survival and degradation mechanism of TX2 under high concentration TC pressure, and evaluating its potential for bioremediation applications in various environments. This study not only provides new bacterial resources for the bioremediation of antibiotic-contaminated environments, but also provides new insights into the biodegradation mechanism of antibiotics.
TC has received widespread attention due to the adverse effects caused by its residues in the environment. At present, most strains isolated from different environments are difficult to degrade high concentrations of TC and apply in practical environments. In addition, previous studies on TC biodegradation of strains have mainly focused on the isolation and characterization of TC-degrading microorganisms, and the characterization of TC degradation mechanisms and the toxicity of their products is relatively limited. Since researchers lack a consensus on the exact mechanism of bacterial degradation of TC, the TC degradation pathway and related genes remain to be elucidated.
The study isolated strain TX2 from the intestine of black soldier fly larvae. The strain can grow in a medium containing 1500 mg/L TC and degrade 400 mg/L TC within 48 h, with a degradation rate of 72.17%. Based on its morphological characteristics, 16S rRNA gene and whole genome phylogenetic analysis, TX2 was identified as Providencia stuartii.
The TC degradation rate of the TX2-inoculated group was significantly higher than that of the CK group, and the difference between the two was mainly attributed to the biodegradation of TX2. In addition, the strain grew well throughout the degradation process, and the pH of the solution increased from 7.0 to 8.4. The reason for the increase in pH may be that bacterial cells decompose peptone in the reaction medium, releasing alkaline compounds such as ammonia and amines, or the degradation of TC may lead to the generation of alkaline substances.
By studying the effects of initial temperature, pH, inoculation amount and initial TC concentration on TC degradation by TX2, it was found that temperature and pH had a significant effect on the biodegradation of TC. At 30℃ and 35℃, the average degradation rates of TC reached 62.51% and 71.25%, respectively, which were significantly higher than the TC degradation rates at 20℃ and 25℃ (Figure 3a); in the initial pH range of 5-9, the TC degradation rates were 55.89%, 57.14%, 66%, 62.55% and 64.44%, respectively. The results showed that higher temperature (30-35℃) and neutral to weakly alkaline conditions were conducive to TX2 degradation of TC. The biodegradation of TC by TX2 was positively correlated with the initial bacterial inoculation amount. The degradation rate of TX2 for 50-200 mg/L TC reached 62.65-97.37%, indicating that TX2 has high degradation efficiency for different concentrations of TC.
A total of 26 potential TC biodegradation intermediates were found. Combined with existing literature reports, the TC degradation pathway of strain TX2 was inferred as shown in the figure, among which pathways II and III are new degradation pathways. In order to evaluate the biological toxicity of TC metabolites after TX2 degradation, ECOSAR toxicity prediction was performed. The results showed that the biodegradation of strain TX2 reduced the toxicity of TC to fish, green algae, and water fleas; the antibacterial activity of the product further confirmed that strain TX2 can reduce the biological toxicity of TC.
The whole genome sequencing results showed that the TX2 strain consists of a circular chromosome with a GC content of 41.14%. The KEGG functional annotation results showed that genes related to exogenous biodegradation, carbohydrate metabolism, and antimicrobial resistance may be involved in the degradation of TC. A total of 10 major antibiotic resistance genes, including tetracyclines, macrolides, and fluoroquinolones, were found in the chromosome of TX2, including 20 TC resistance genes and 43 multidrug resistance genes. In addition, laccase and glutathione-S transferase genes were detected on the genome of TX2. The presence of these genes is the key to TX2's degradation of TC. Among the six known TC-degrading enzymes, only GST was detected at the transcriptome level and the expression level was only upregulated by 1.97 times. However, this is not enough to explain TX2's efficient TC degradation ability.
Therefore, this study combined multi-omics analysis to clarify the three mechanisms of TX2 resistance and degradation to TC: i) TC is pumped out of the cell through the efflux pump family (MdtA, MdtB, Mate family and Tet (B) etc.); ii) TC is degraded by degradative enzymes, including glutathione-S transferase, oxygenase, esterase, oxidoreductase, fumaryl acetate hydrolase family, superoxide dismutase etc.; iii) The role of broad-spectrum stress kinases and antimicrobial resistance enzymes and molecular chaperones that assist in the correct folding of proteins under adversity. Interestingly, the upregulation of typical ribosome protection mechanism proteins (elongation factors Tu and G etc.) was not significant. In addition, we found that the expression levels of a large number of proteins with unknown functions were upregulated under TC pressure. We speculate that the unknown functional proteins may include new genes involved in the ribosome protection mechanism and other novel degradation enzyme genes.
Compared with the control group, the degradation rates of TC or CTC in chicken manure (CM), soil (SL), water (WW) and chlortetracycline residue (CFR) in the TX2 group were increased by 18.36%, 21.63%, 20.28% and 32.05%, respectively. In addition, the immobilized IMP-TX2 was able to operate stably in the WW system for at least 35 days (5 cycles). The above results show that the addition of TX2 significantly promotes the degradation of tetracycline antibiotics in the environment, especially in the treatment of TC-contaminated wastewater.
In addition, in order to further evaluate the ecological safety of TX2 application, samples with high tetracycline resistance gene (TRGs) abundance (CM) and low TRGs abundance (SL) were selected, and the abundance of 8 TRGs (tetA, tetC, tetG, tetL, tetM, tetO, tetW and tetX) and the integrase gene intI-1 before and after TX2 bioaugmentation were identified. The results showed that in the CM group, the abundance of the above genes decreased to varying degrees after bioaugmentation with TX2; while in the SL group, the addition of TX2 had little effect on the abundance of TRGs. The above results indicate that TX2 exhibits a stronger remediation effect in an environment with high initial TRGs levels, while it has little disturbance on the abundance of ARGs and MGEs in an environment with low initial TRGs levels. These findings highlight the potential application of TX2 in removing TC pollution.
In this study, a new type of efficient TC-degrading bacterium TX2 was isolated from the intestine of the saprophytic insect BSFL, which tolerates high concentrations of TC, and was identified as Providencia stuartii. It can effectively degrade TC with an initial concentration of 400 mg/L by 72.17% within 48 hours. TX2 metabolizes TC into 27 potential degradation products through processes such as isomerization, hydroxylation, oxygenation, ring opening and degrouping, and significantly reduces the toxicity of TC. Multi-omics joint analysis revealed the key enzymes and mechanisms of TX2 in degrading TC. In addition, its bioaugmentation application in CM of samples with high ARGs contamination not only significantly reduced TC concentrations, but also effectively reduced the incidence of ARGs and MGEs. This study provides new insights into the biodegradation mechanism of antibiotics and provides valuable microbial resources for bioaugmentation technology for antibiotic removal.
