Establishment of an axenic culture of microalgae Pectinodesmus pectinatus and genome-based antibiotic resistance profiling of its associated bacteria

다국어 초록 (Multilingual Abstract)

Freshwater microalgae–bacteria consortia are increasingly used in wastewater treatment and biomass production, yet their bacterial partners may serve as reservoirs of multidrug-resistant (MDR) bacteria and antibiotic resistance genes (ARGs). In this study, we isolated three bacterial strains (Chryseobacterium sp. P1, Pseudomonas monteilii P2, and Stenotrophomonas pavanii P3) from the phycosphere of the freshwater microalga Pectinodesmus pectinatus and characterized their genomic and phenotypic traits related to antibiotic resistance. Whole-genome sequencing and comparative genomic analyses were combined with minimum inhibitory concentration (MIC) testing against 16 antibiotics. ARGs were predicted using multiple databases and mapped onto functional modules, including L-Ara4N/PEtN-mediated lipid A modification, lipopolysaccharide (LPS) biosynthesis and transport, efflux pumps, and putative two-component regulatory systems. All three isolates carried diverse ARGs and exhibited elevated MICs to several β-lactams; colistin MICs differed among isolates (≥16 μg/mL in Chryseobacterium sp. P1 and S. pavanii P3 vs ≤2 μg/mL in P. monteilii P2), and S. pavanii showed the broadest MDR phenotype. Consistent with this phenotype, it harbored the most extensive repertoire of genes associated with L- Ara4N/PEtN modification (e.g. arn family, eptA), LPS/outer-membrane maintenance, and multidrug efflux that have been implicated in, and may contribute to, colistin non- susceptibility. Qualitative genotype–phenotype concordance analysis across three bacterial isolates revealed that many resistance patterns could be explained by the presence or absence of known determinants. Overall, our results suggest that the P. pectinatus phycosphere can harbor MDR and colistin-non-susceptible Stenotrophomonas strains and underscore freshwater microalgae-based systems as potential environmental reservoirs of clinically relevant antibiotic resistance.

목차 (Table of Contents)

• Ⅰ. Introduction 7
• Ⅱ. Materials and Methods 11
• 1. Acquisition and maintenance of ten microalgal strains 11
• 2. Cleaning of P. pectinatus cultures using NaClO 13
• 3. Isolation of phycosphere-associated bacteria from P. pectinatus 14
• 4. Whole genome sequencing, genome assembly, and annotation 15
• 5. Antimicrobial susceptibility tests of three bacterial isolates 17
• 6. 16S rRNA gene–based phylogenetic analysis 19
• 7. Genome-based taxonomic analysis 20
• 8. In silico prediction of antibiotic resistance genes 21
• 9. Identification of colistin-associated chromosomal determinants 23
• Ⅲ. Results 25
• 1. Axenic culture status of ten freshwater green microalgal strains 25
• 2. Effect of NaClO-based cleaning on the cell surface of P. pectinatus 26
• 3. Isolation and colony morphology of three bacterial strains from P. pectinatus 28
• 4. 16S rRNA gene–based phylogenetic placement of three bacterial isolates 30
• 5. Assembly statistics and general genomic features of three bacterial isolates 33
• 6. MIC profiles of three isolates against a panel of 16 antibiotics 35
• 7. Whole-genome architecture and chromosomal distribution of antibiotic resistance genes in
• three isolates 37
• 8. WGS-based resistome comparison revealing strain-specific resistance breadth 42
• 9. Qualitative concordance between WGS-predicted resistomes and MIC profiles in three
• isolates 45
• 10. Comparative distribution of colistin-associated chromosomal determinants in three
• isolates 48
• Ⅳ. Discussion 51
• Ⅴ. 국문초록 65
• Acknowledgement 67
• Reference 68