Abstract: Bifidobacterium longum is a commonly used probiotic in food industry, and its drug resistance is related to food safety issues. The objective of this study was to formulate antibiotic resistance determination criteria for B. longum and to explore the key genes of antibiotic resistance through functional gene analysis. The minimum inhibitory concentrations of 52 B. longum strains towards tetracycline, erythromycin, clindamycin, ampicillin, chloramphenicol, and vancomycin were determined with reference to ISO standard methods (ISO 10932). The species-specific microbiological cut-off values of these six antibiotics of B. longum were evaluated by Turnidge (T) and Kronvall (K) statistical methods and analyzed the antibiotic resistance mechanism from the molecular genetic basis. The results showed that the species-specific microbiological cut-off values for tetracycline, erythromycin, clindamycin, ampicillin, chloramphenicol, and vancomycin of B. longum were 8, 8, 0.25, 8/2(T/K), 8 and 2 μg/mL respectively, and the drug resistance rates were 28.85%, 25%, 28.85%, 3.85%/7.69%(T/K), 0 and 19.23%. Functional gene and comparative genomic analysis revealed that tet(W) and erm(X) both located on mobile genetic elements were the main resistance genes for tetracycline resistance and erythromycin and clindamycin resistancein B.longum.Phylogeneticanalysis indicated that the tet(W) and erm(X) gene sequences of different phenotypic resistance strains possessed significant variation. This study has certain guiding significance and reference value for the safety application of B. longum in food and the development of evaluation criteria for probiotics resistance.
[1] O′CALLAGHAN A,VAN S D.Bifidobacteria and their role as members of the human gut microbiota[J].Frontiers in Microbiology,2016,7:925. [2] 丁喜顺.长双歧杆菌细胞壁完整肽聚糖的分离及其免疫、抑瘤活性的研究[D].呼和浩特:内蒙古农业大学,2009. [3] 闫爽.长双歧杆菌遗传与表型多样性及其与免疫调节功能的相关性研究[D].无锡:江南大学,2019. [4] JANINA M,ANNETTE H,CLAUDIUS M,et al.Efficacy of Bifidobacterium longum, B. infantis and Lactobacillus acidophilus probiotics to prevent gut dysbiosis in preterm infants of 28+0-32+6 weeks of gestation: a randomised, placebo-controlled, double-blind, multicentre trial: the PRIMAL clinical study protocol[J].BMJ Open,2019,9(11):e032617. [5] INTURRI R,TROVATO L,VOLTI G L,et al.In vitro inhibitory activity of Bifidobacterium longum BB536 and Lactobacillus rhamnosus HN001 alone or in combination against bacterial and Candida reference strains and clinical isolates[J].Heliyon,2019,5(11):e02891. [6] MIJOO C,YUNJUNG L,NA-KYOUNG L,et al.Immunomodulatory effects by Bifidobacterium longum KACC 91563 in mouse splenocytes and macrophages[J].Journal of Microbiology and Biotechnology,2019,29(11):1 739-1 744. [7] 丁圣,蒋菁莉,刘松玲,等.长双歧杆菌BBMN68对便秘模型小鼠的通便作用[J].食品科学,2011,32(3):195-198. [8] XIAO J Z,KONDO S,TAKAHASHI N,et al.Effects of milk products fermented by Bifidobacterium longum on blood lipids in rats and healthy adult male volunteers[J].Journal of Dairy Science,2003,86(7):2 452-2 461. [9] OTHMAN M B,SAKAMOTO K.Effect of inactivated Bifidobacterium longum intake on obese diabetes model mice (TSOD)[J].Food Research International,2020,129(108 792). [10] FAHMY C A,GAMAL-ELDEEN A M, EL-HUSSIENY E A,et al.Bifidobacterium longum suppresses murine colorectal cancer through the modulation of oncomi.Rs and tumor suppressor miRNAs[J].Nutrition and Cancer,2019,71(4):688-700. [11] EFSA BIOHAZ Panel (EFSA Panel on Biological Hazards).Scientific opinion on the maintenance of the list of QPS biological agents intentionally added to food and feed (2013 update)[J].EFSA Journal,2013,11(11):3 449. [12] 潘伟好,李平兰,孙承虎.双歧杆菌的药敏性及其质粒DNA的检测[J].食品科学,2005,26(6):81-86. [13] DURANTI S,LUGLI G A,MANCABELLI L,et al.Prevalence of antibiotic resistance genes among human gut-derived bifidobacteria[J].Applied and Environmental Microbiology,2016,83(3):e02 894-16. [14] FOUHY F,O’CONNELL M M,FITZGERALD G F,et al.In silico assigned resistance genes confer Bifidobacterium with partial resistance to aminoglycosides but not to β-lactams[J].PloS One,2013,8(12):e82653. [15] 罗义,周启星.抗生素抗性基因(ARGs)——一种新型环境污染物[J].环境科学学报,2008,28(8):1 499-1 505. [16] European Food Safety Authority (EFSA).Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance[J].EFSA Journal,2012,10(6):2 740. [17] European Committee on Antimicrobial Susceptibility Testing.MIC distributions and epidemiological cut-off value (ECOFF) setting[P].EUCAST SOP 10.0,2017.http://www.eucast.org. [18] KLARE I, KONSTABEL C, WERNER G,et al.Antimicrobial susceptibilities of Lactobacillus, Pediococcus and Lactococcus human isolates and cultures intended for probiotic or nutritional use[J].Journal of Antimicrobial Chemotherapy,2007,59(5):900-912. [19] International Stardand Organization (ISO).Milk and milk products-determination of the minimal inhibitory concentration (MIC) of antibiotics applicable to bifidobacteria and non-enterococcal lactic acid bacteria (LAB)[S].ISO,2010. [20] TURNIDGE J,KAHLMETER G,KRONVALL G.Statistical characterisation of bacterial wild-type MIC value distributions and the determination of microbiological cut-off values[J].Clinical Microbiology and Infection,2006,12(5):418-425. [21] KRONVALL G.Normalized resistance interpretation as a tool for establishing epidemiological MIC susceptibility breakpoints[J].Journal of Clinical Microbiology,2010,48(12):4 445-4 452. [22] European Committee on Antimicrobial Susceptibility Testing (EUCAST). EUCAST general consultation on “considerations in the numerical estimation of epidemiological cutoff (ECOFF) values”[J].EUCAST,2018. [23] CAMPEDELLI I,MATHUR H,SALVETTI E,et al.Genus-wide assessment of antibiotic resistance in Lactobacillus spp.[J].Applied and Environmental Microbiology,2019,85(1):e01 738-18. [24] HU Y,YANG X,LI J,et al.The bacterial mobile resistome transfer network connecting the animal and human microbiomes[J].Applied and Environmental Microbiology,2016,82(22):6 672-6 681. [25] 迟星云,朱晓莉,王虹,等.重症监护病房多重耐药菌检出及药物敏感性[J].中华医院感染学杂志,2019,29(24):3 729-3 733. [26] MARTíNEZ N,LUQUE R,MILANI C,et al.A gene homologous to rRNA methylase genes confers erythromycin and clindamycin resistance in Bifidobacterium breve[J]. Applied and Environmental Microbiology,2018,84(10):e02 888-17. [27] WEI Y X.Safety assessment of Bifidobacterium longumJDM301 based on complete genome sequences[J].World Journal of Gastroenterology,2012,18(5):479-488.
2020, Vol. 46 
