市售酸奶中乳酸菌的鉴定与耐药性

Identification of lactic acid bacteria in commercial yogurt and their antibiotic resistance

摘要:

摘要:【目的】检测市售酸奶中乳酸菌的种类及其耐药情况。【方法】收集10种来自5个不同厂家的酸奶，通过细菌基因组重复基因外回文序列-PCR(repetitive extragenic palindromic-PCR，rep-PCR)结合16S rRNA同源性分析的方法对分离的乳酸菌进行基因分型和菌种鉴定。利用药敏纸片扩散法(K-B 法)对分离的乳酸菌进行针对7种抗生素的药敏实验，用PCR 特异性扩增结合测序的方法检测每个样品中不同基因型菌株的耐药基因(包括红霉素耐药基因erm A、erm B 和四环素耐药基因tet M、tet K、tet S、tet Q、tet O、tet L、tet W)。【结果】10 种市售酸奶中分离到100株乳酸菌。其中，德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckii ssp．bulgaricus)23 株，干酪乳杆菌( Lactobacillus casei)26株，嗜热链球菌(Streptococcus thermophilus)30株，嗜酸乳杆菌(Lactobacillus acidophilus) 5株，植物乳杆菌(Lactobacillus plantarum)6株，副干酪乳杆菌(Lactobacillus paracasei)10株。药敏实验发现所有100株乳酸菌均对链霉素和庆大霉素耐药，42株对万古霉素耐药，没有菌株对头孢氨苄，四环素，红霉素以及土霉素耐药。在28株经过16S rRNA测序的乳酸菌中检测到5种不同的耐药基因，在8株乳酸菌中检测到erm B基因，4株检测到tet K基因，2株菌检测到tet L基因，4株菌检测 到tet M基因，2株菌检测到tet O基因，没有检测到erm A，tet S，tet Q，tet W基因。28株乳酸菌中有15株(53.57% )检测到耐药基因，其中有4株L.delbrueckii ssp．bulgaricus 检测到2－3种不同的耐药基因。【结论】本研究在市售酸奶中除了检测到商品标签上标注的L.delbrueckii ssp. bulgaricus和S.thermophilus以外，还检测到商标上没有标注的乳酸菌;作为常用发酵剂的德氏乳杆菌保加利亚亚种和嗜热链球菌更容易检测到耐药基因;分离得到的乳酸菌均对红霉素和四环素敏感却检测到相应的耐药基因，再一次证明了没有耐药表型的菌株也可能携带耐药基因。

Abstract:

Abstract:［Objective］ To identify lactic acid bacteria (LAB) in commercial yogurts and investigate their antibiotic resistance.［Methods］LABs were cultured from 5 yogurt brands and the isolates were identified at the species level by 16S rRNA sequence． Genotyping was performed by repetitive extragenic palindromic PCR (rep-PCR)．The sensitivity to 7 antibiotics was tested for all LAB isolates by Kirby-Bauer paper diffusion (K-B method). Meanwhile，9 antibiotic resistance genes(ARGs)，including erythromycin resistance genes (ermA and ermB) and tetracycline resistance genes (tetM，tetK，tetS，tetQ，tetO，tetL and tetW)，were detected by PCR amplification in the identified LAB isolates．The PCR products were confirmed by sequencing． ［Results］ Total 100 LABs were isolated，including 23 Lactobacillus delbrueckii ssp．bulgaricus，26 Lactobacillus casei，30 Streptococcus thermophilus，5 Lactobacillus acidophilus，6 Lactobacillus plantarum，and 10 Lactobacillus paracasei. The drug susceptibility test shows that all 100 isolates were resistant to gentamicin and streptomycin，42 isolates were resistant to vancomycin，and on the contrary all were sensitive to cefalexin，erythromycin，tetracycline and oxytetracycline. Moreover，5 ARGs were found in the 28 sequencing confirmed isolates，ermB gene was detected in 8 isolates，tet K in 4 isolates，tetL in 2 isolates，tetM in 4 isolates，tetO in 2 isolates. erm A，tet S，tet Q and tet W genes were not detected in the isolates． Antibiotic resistance genes were found in 53.57% (15/28) sequenced isolates，2－3 antibiotic resistance genes were detected in 4 isolates of L.delbrueckii ssp． bulgaricus.［Conclusions］Some LABs were not labeled in commercial yogurt products. Antibiotic resistance genes tend to be found in the starter culture of L. delbrueckii ssp． Bulgaricus and S. thermophilus. All the LAB isolates were sensitive to erythromycin and tetracycline，even though some carried erythromycin and/or tetracycline resistance genes．We proved again that LAB could carry antibiotic resistance gene(s) though it is sensitive to antibiotics．