Pantoea eucrina B1-6 之生物膜形成與抗藥性特性

Abstract

抗生素的濫用已被世界衛生組織列為全球公共衛生的重大威脅，其核心主因在於多重抗藥性菌株的迅速蔓延，因此深入釐清微生物對抗生素的耐受與防禦機制至關重要。細菌生物膜不僅是臨床院內感染的主要根源，亦是微生物阻絕藥物、提升抗藥性的關鍵原因。本研究針對先前自小黃瓜分離且具強烈黏液表型之菌株 Pantoea eucrina B1-6 進行探討。將全基因體定序資料，利用 CARD 與 DFAST 資料庫進行生物資訊學分析，系統性鑑定出多種與藥物外排相關之幫浦及調控基因，並於基因組 contig 2 上發現結構完整之水平轉移元件及其攜帶之 MFS 轉運蛋白 (MFS transporter)。在抗生素敏感性實驗中，P. eucrina B1-6 對於 norfloxacin 與 azithromycin 抗生素之耐受性高於對照組 E. coli K12。為評估生物膜型態對藥物清除的影響，本研究進一步進行生物膜清除實驗。結果顯示，經 1 至 8 倍 MIC 濃度的 norfloxacin 處理後，生物膜細胞活性反而較對照組提升接近 1.5 倍；而 azithromycin 雖能有效抑制細胞活性，但在高濃度下則出現活性回升現象。值得注意的是，在兩種抗生素壓力下，生物膜內的活菌數與對照組相比均無顯著差異，證實微生物一旦形成生物膜，抗生素對其存活菌量的殺滅與清除能力將大幅下降，印證了生物膜屏障與高耐受特性。最後，透過 RT-qPCR 深入解析生物膜之分子機制。結果證實，施加 0.5 及 1 µg / mL 的 norfloxacin 會顯著抑制 RNA 結合蛋白基因 rsmA 的表現，並大幅誘導 recA 的表現，顯示菌株正積極啟動應變反應以維護遺傳物質之完整性；相反地，在 512 µg / mL 的高濃度 azithromycin 處理下，多重抗藥外排幫浦 kpnH 及全球性轉錄調節因子 crp 的表現量同步提升，表示菌株正透過基因調控來加速藥物外排。本研究全面剖析了 P. eucrina B1-6 的基因組特徵與生物膜抗藥表型，並探究其分子機制，期望能為未來生物膜臨床治療與公共衛生防禦，提供理論基礎。

The abuse of antibiotics has been recognized by the World Health Organization as a major threat to global public health, primarily driven by the rapid dissemination of multidrug-resistant strains. Therefore, it is crucial to thoroughly elucidate the resistance and defense mechanisms of microorganisms against antibiotics. Bacterial biofilms are not only a primary source of nosocomial infections in clinical settings but also a key factor through which microorganisms limit antibiotic penetration and enhance resistance. This study focuses on Pantoea eucrina B1-6, a strain previously isolated from cucumbers that exhibits a strong mucoid phenotype. By performing bioinformatics analysis on whole-genome sequencing data using the CARD and DFAST databases, multiple efflux pumps and regulatory genes associated with drug efflux were systematically identified. Furthermore, a structurally complete horizontal gene transfer element carrying an MFS transporter was discovered on genome contig 2. In antimicrobial susceptibility testing, P. eucrina B1-6 demonstrated higher tolerance toward fluoroquinolones and macrolides (norfloxacin and azithromycin) than the control strain E. coli K12. To evaluate the impact of biofilm morphology on drug eradication, biofilm eradication assays were performed. The results indicated that treatment with 1 to 8 fold MIC of norfloxacin unexpectedly increased biofilm cell activity by nearly 1.5 fold compared to the control group. Conversely, while azithromycin effectively suppressed initial cell viability, a rebound in activity was observed at higher concentrations. Notably, under the stress of both antibiotics, the viable cell counts within the biofilms showed no significant difference compared to the control group. This confirms that once microorganisms form biofilms, the bactericidal and eradication efficacy of antibiotics against viable cells drops drastically, demonstrating the barrier effects and high tolerance characteristics of biofilms. Finally, RT-qPCR was utilized to gain deeper insights into the molecular mechanisms of the biofilms. The results demonstrated that the application of 0.5 and 1 µg / mL of norfloxacin significantly inhibited the expression of the RNA binding protein gene rsmA while substantially inducing the expression of recA, indicating that the strain actively initiates a stress response to maintain genome integrity. In contrast, under treatment with a high concentration of azithromycin (512 µg / mL), the expression levels of the multidrug efflux pump kpnH and global transcriptional regulator crp were simultaneously upregulated, demonstrating that the strain accelerates drug efflux through gene regulation. This study provides a comprehensive analysis of the genomic features and biofilm resistance phenotypes of P. eucrina B1-6 and explores its underlying molecular mechanisms, aiming to provide a solid and critical scientific foundation for future clinical treatment of biofilms and public health defense.