该研究旨在通过实验室进化的方法提高植物乳杆菌对头孢氨苄的耐药性,并对耐药性植物乳杆菌的益生性状进行评价,同时应用全基因组测序技术检测进化菌株的基因突变。植物乳杆菌在头孢氨苄存在下进化时,最低抑制浓度(minimum inhibitory concentration,MIC)由起始的16 μg/mL提高到8 192 μg/mL,耐药性提高了512倍,当在去除头孢氨苄后进一步进化时,其MIC值由8 192 μg/mL降低至4 096 μg/mL,但在此后进化中保持稳定,与原始菌株相比,其耐药性提高了256倍,表明通过实验室进化可获得具有高耐药性的植物乳杆菌。对高耐药性植物乳杆菌益生性状的研究表明,植物乳杆菌在进化后其菌株的耐酸,耐胆盐能力和黏附能力没有发生明显改变,表明进化菌株的益生性状稳定。菌株的全基因组测序也检测到与耐药性和益生性状相关的突变基因。研究结果为植物乳杆菌与抗生素的联合使用提供了参考。
This study aimed to improve the resistance of Lactobacillus plantarum to cephalexin by laboratory evolution, and to evaluate the probiotic properties of resistant strains. At the same time, whole-genome sequencing was used to detect gene mutations of evolved strains. When L. plantarum evolved in the presence of cephalexin, its minimum inhibitory concentration (MIC) increased from the original 16 μg/mL to 8 192 μg/mL, and the resistance increased by 512 times. When cephalexin was removed and L. plantarum further evolved, its MIC value reduced from 8 192 μg/mL to 4 096 μg/mL, but it remained stable during the subsequent evolution. Compared with the original strain, its resistance increased by 256 times, indicating that L. plantarum with high resistance could be obtained through laboratory evolution. The study on the probiotic properties of highly resistant L. plantarum showed that the acid tolerance, bile salts tolerance and adhesion ability of the strains did not change significantly after evolution, indicating that probiotic properties of the evolved strain were stable. Whole genome sequencing of the strains also detected mutant genes related to resistance and probiotic properties. The results provide a reference for the combined use of L. plantarum and antibiotics.
[1] 王永红, 冉茂娟, 龙晓莉.莫西沙星联合头孢氨苄治疗老年患者下呼吸道细菌感染的临床疗效[J].中华医院感染学杂志, 2015,25(3):572-574.
WANG Y H, RAN M J, LONG X L.The clinical effect of moxifloxacin combined cefalexin in the treatment of lower respiratory tract bacterial infections in elderly patients[J].Chinese Journal of Nosocomiology, 2015,25(3):572-574.
[2] ZAMAN S B, HUSSAIN M A, NYE R, et al.A review on antibiotic resistance:Alarm bells are ringing[J].Cureus, 2017, 9(6):e1403.
[3] MARCO M L, PAVAN S, KLEEREBEZEM M.Towards understanding molecular modes of probiotic action[J].Current Opinion in Biotechnology, 2006, 17(2):204-210.
[4] PREIDIS G A, VERSALOVIC J.Targeting the human microbiome with antibiotics, probiotics, and prebiotics:Gastroenterology enters the metagenomics era[J].Gastroenterology, 2009, 136(6):2 015-2 031.
[5] BANERJEE P, MERKEl G J, BHUNIA A K.Lactobacillus delbrueckii ssp.bulgaricus B-30892 can inhibit cytotoxic effects and adhesion of pathogenic Clostridium difficile to Caco-2 cells[J].Gut Pathogens, 2009, 1(1):8.
[6] DE VRIES M C, VAUGHAN E E, KLEEREBEZEM M, et al.Lactobacillus plantarum-survival, functional and potential probiotic properties in the human intestinal tract[J].International Dairy Journal, 2006, 16(9):1 018-1 028.
[7] GUEIMONDE M, SÁNCHEZ B, DE LOS REYES-GAVILÁN C G, et al.Antibiotic resistance in probiotic bacteria[J].Frontiers in Microbiology, 2013, 4:202.
[8] HORINOUCHI T, FURUSAWA C.Understanding metabolic adaptation by using bacterial laboratory evolution and trans-omics analysis[J].Biophysical Reviews, 2020, 12(3):677-682.
[9] GENEVA I O F S, BRUSSELS I D F.Milk and milk products.Determination of the minimal inhibitory concentration (MIC) of antibiotics applicable to bifidobacteria and non-enterococcal lactic acid bacteria (LAB)[S].2010.
[10] LÁZÁR V, PAL SINGH G, SPOHN R, et al.Bacterial evolution of antibiotic hypersensitivity[J].Molecular Systems Biology, 2013, 9(1):700.
[11] SUI L, ZHU X M, WU D Y, et al.In vitro assessment of probiotic and functional properties of Bacillus coagulans T242[J].Food Bioscience, 2020, 36(1):100675.
[12] PATEL A K, AHIRE J J, PAWAR S P, et al.Comparative accounts of probiotic characteristics of Bacillus spp.isolated from food wastes[J].Food Research International, 2009, 42(4):505-510.
[13] COLLADO M C, MERILUOTO J, SALMINEN S.Adhesion and aggregation properties of probiotic and pathogen strains[J].European Food Research and Technology, 2008, 226(5):1 065-1 073.
[14] HUANG R H, TAO X Y, WAN C X, et al.In vitro probiotic characteristics of Lactobacillus plantarum ZDY 2013 and its modulatory effect on gut microbiota of mice[J].Journal of Dairy Science, 2015, 98(9):5 850-5 861.
[15] FENG C J, ZHANG F X, WANG B N, et al.Genome-wide analysis of fermentation and probiotic trait stability in Lactobacillus plantarum during continuous culture[J].Journal of Dairy Science, 2020, 103(1):117-127.
[16] JANG S, KIM M, HWANG J, et al.Tools and systems for evolutionary engineering of biomolecules and microorganisms[J].Journal of Industrial Microbiology & Biotechnology, 2019, 46(9-10):1 313-1 326.
[17] ARNOLD F H, WINTRODE P L, MIYAZAKI K, et al.How enzymes adapt:Lessons from directed evolution[J].Trends in Biochemical Sciences, 2001, 26(2):100-106.
[18] SOLEYMANZADEH MOGHADAM S, KHODAII Z, FATHI ZADEH S, et al.Synergistic or antagonistic effects of probiotics and antibiotics-alone or in combination-on antimicrobial-resistant Pseudomonas aeruginosa isolated from burn wounds[J].Archives of Clinical Infectious Diseases, 2018, 13(3):e63121.
[19] GEORGE J, HALAMI P M.Subinhibitory concentrations of gentamicin triggers the expression of aac (6′)Ie- aph (2″)Ia, chaperons and biofilm related genes in Lactobacillus plantarum MCC 3011[J].Research in Microbiology, 2017, 168(8):722-731.
[20] DONG Y S, ZHANG F X, WANG B N, et al.Laboratory evolution assays and whole-genome sequencing for the development and safety evaluation of Lactobacillus plantarum with stable resistance to gentamicin[J].Frontiers in Microbiology, 2019, 10:1 235.
[21] FLUMAN N, BIBI E T.Bacterial multidrug transport through the lens of the major facilitator superfamily[J].Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 2009, 1 794(5):738-747.
[22] SEKIGUCHI J I, MIYOSHI-AKIYAMA T, AUGUSTYNOWICZ-KOPEC′ E, et al.Detection of multidrug resistance in Mycobacterium tuberculosis[J].Journal of Clinical Microbiology, 2007, 45(1):179-192.
[23] NCBI (National Center for Biotechnology Information).prsA2-Foldase protein PrsA 2 precursor-Streptococcus pyogenes serotype M1-prsA2 gene & protein[DB].2021.
[24] NCBI (National Center for Biotechnology Information).CDD Conserved Protein Domain Family:YibE-F[DB].Accessed Nov.19, 2015.
[25] GUO L, WU T, HU W, et al.Phenotypic characterization of the foldase homologue PrsA in Streptococcus mutans[J].Molecular Oral Microbiology, 2013, 28(2):154-165.
