The development of variation-based rifampicin resistance in Staphylococcus aureus deciphered through genomic and transcriptomic study.

Rifampicin (RIF) resistance imposes a challenge on the antimicrobial treatment of pathogen infections. Figuring out the development mechanism of RIF resistance is critical to improving antimicrobial therapy strategy in clinics and biological treatment strategy of RIF polluted sewage in environmental engineering. The RIF resistance development of Staphylococcus aureus (S. aureus) with exposure to RIF at sub-inhibitory concentrations was comprehensively investigated via genomic and transcriptomic approaches in this study. RIF minimal inhibitory concentration (MIC) for S. aureus rapidly increased from 0.032 to 256 mg/L. Membrane permeability decrease, biofilm formation enhancement, and ROS production increase associated with RIF resistance were observed in RIF-induced strains. Through comparative genomic analysis, mutations in rpoB and rpoC were considered to be associated with RIF resistance in S. aureus mutants. Pan-genome-wide single-nucleotide variant analysis indicated that mutations at rpoB-1412, rpoB-1451, and rpoB-1457 were prevalent in 13849 public genomes of S. aureus, while mutations at rpoB-2256, and rpoC-3092 were first discovered in this study. The panorama of adaptative alteration of cellular physiological processes was observed via transcriptomic analysis. The oxidation pressure responses, metabolism, transporters, virulence factors, and multiple steps of DNA and RNA machinery were found to be perturbed by RIF in S. aureus.

New insights into lincomycin biodegradation by Conexibacter sp. LD01: Genomics characterization, biodegradation kinetics and pathways.

The aerobic, lincomycin-degrading bacterial strain Conexibacter sp. LD01, belonging to the phylum Actinobacteria, was isolated from activated sludge. Both second- and third-generation sequencing technologies were applied to uncover the genomic characterization and high-quality genome with 99.2% completeness and 2.2% contamination was obtained. The biodegradation kinetics of lincomycin fit well with the modified Gompertz model (R2 > 0.97). Conexibacter sp. LD01 could subsist with lincomycin as the sole source of carbon, nitrogen, and energy. When 500 mg/L of glucose was added as a co-substrate, the biodegradation rate improved significantly, whereas the addition of 500 mg/L sodium pyruvate had a slight inhibitory effect. Ammonia nitrogen was the best nitrogen source for Conexibacter sp. LD01 when growing and degrading lincomycin. In total, 17 metabolic products consisting of nine novel products were detected, and five biodegradation pathways, including N-demethylation, breakage of the amido bond, sulfoxidation, and oxidation of the pyrrolidine ring and propylamino chain, were proposed. This study significantly expands our understanding of the functional microorganisms and mechanism involved in lincomycin biodegradation at the phylum level.

Deciphering chloramphenicol biotransformation mechanisms and microbial interactions via integrated multi-omics and cultivation-dependent approaches.

BACKGROUND: As a widely used broad-spectrum antibiotic, chloramphenicol is prone to be released into environments, thus resulting in the disturbance of ecosystem stability as well as the emergence of antibiotic resistance genes. Microbes play a vital role in the decomposition of chloramphenicol in the environment, and the biotransformation processes are especially dependent on synergistic interactions and metabolite exchanges among microbes. Herein, the comprehensive chloramphenicol biotransformation pathway, key metabolic enzymes, and interspecies interactions in an activated sludge-enriched consortium were elucidated using integrated multi-omics and cultivation-based approaches. RESULTS: The initial biotransformation steps were the oxidization at the C1-OH and C3-OH groups, the isomerization at C2, and the acetylation at C3-OH of chloramphenicol. Among them, the isomerization is an entirely new biotransformation pathway of chloramphenicol discovered for the first time. Furthermore, we identified a novel glucose-methanol-choline oxidoreductase responsible for the oxidization of the C3-OH group in Sphingomonas sp. and Caballeronia sp. Moreover, the subsequent biotransformation steps, corresponding catalyzing enzymes, and the microbial players responsible for each step were deciphered. Synergistic interactions between Sphingomonas sp. and Caballeronia sp. or Cupriavidus sp. significantly promoted chloramphenicol mineralization, and the substrate exchange interaction network occurred actively among key microbes. CONCLUSION: This study provides desirable strain and enzyme resources for enhanced bioremediation of chloramphenicol-contaminated hotspot sites such as pharmaceutical wastewater and livestock and poultry wastewater. The in-depth understanding of the chloramphenicol biotransformation mechanisms and microbial interactions will not only guide the bioremediation of organic pollutants but also provide valuable knowledge for environmental microbiology and biotechnological exploitation. Video Abstract.

New insights into thiamphenicol biodegradation mechanism by Sphingomonas sp. CL5.1 deciphered through metabolic and proteomic analysis.

Biological treatment is an efficient and economical process to remove thiamphenicol (TAP) residues from the environment. The discovery of TAP-degrading bacteria and the decryption of its biodegradation mechanism will be beneficial to enhance the biological removal of TAP. In this study, Sphingomonas sp. CL5.1 was found to be capable of catabolizing TAP as the sole carbon, nitrogen, and energy source. This strain could degrade 93.9% of 25 mg/L TAP in 36 h, and remove about 11.9% of the total organic carbon of TAP. A novel metabolism pathway of TAP was constructed, and the enzymes involved in TAP metabolism in strain CL5.1 were predicted via proteomic and metabolic analysis. TAP was proposed to be transformed to O-TAP via oxidation of C3-OH and DD-TAP via dehydration of C3-OH and dehydrogenation of C1-OH. A novel glucose-methanol-choline (GMC) family oxidoreductase CapO was predicted to be involved in the oxidation of C3-OH. O-TAP was supposed to be further cleaved into DCA, glycine, and PMB. Glycine might be a pivotal direct nitrogen source for strain CL5.1, and it could be involved in nitrogen metabolism through the glycine cleavage system or directly participate in the biosynthetic processes.

Chloramphenicol biodegradation by enriched bacterial consortia and isolated strain Sphingomonas sp. CL5.1: The reconstruction of a novel biodegradation pathway.

Figuring out the comprehensive metabolic mechanism of chloramphenicol (CAP) is critical to improving CAP removal in the bioremediation process. In this study, CAP biodegradation by six consortia and isolated Sphingomonas sp. CL5.1 were systematically investigated using the combination of high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry, second-generation, and third-generation sequencing technologies. The CAP-degrading capability of six consortia was enhanced while CAP mineralization rate declined after long-term enrichment. The microbial community structures of six consortia were all simplified with 69%-82% decline in species richness after continuous passages for one year. The core genera of consortia CL and CH included Sphingomonas, Cupriavidus, Burkholderia, Chryseobacterium, and Pigmentiphaga, which accounted for over 98% of the total population. Sphingomonas was discovered as a new CAP degrader that could subsist on CAP as the sole carbon, nitrogen, and energy sources. Sphingomonas sp. CL5.1 was able to completely remove 120 mg/L CAP within 48 hours with a mineralization rate of 50.4%. The presence of acetate or nitrite could inhibit CAP metabolization by strain CL5.1. Four CAP metabolic pathways were constructed, including modification of the C3 hydroxyl group of CAP via acetylation, oxidization, dehydration and the bond cleavage between C1 and C2. C3 hydroxyl group dehydration and C1-C2 bond-cleavage were first reported regarding to CAP biotransformation. Strain CL5.1 played a core role in the consortia and was responsible for C3 hydroxyl oxidation, C3 dehydration, and C1-C2 bond cleavage. Genomic information of strain CL5.1 revealed the further mineralization pathways of downstream product p-nitrobenzoic acid via ortho- and meta-cleavage.