Moodley Clinton, Reid Sharon J, Abratt Valerie R
Department of Molecular and Cell Biology, University of Cape Town, Cape Town, South Africa.
Anaerobe. 2015 Apr;32:63-69. doi: 10.1016/j.anaerobe.2014.12.004. Epub 2014 Dec 18.
Administration of probiotic bacteria such as Bifidobacterium spp. can prevent antibiotic associated diarrhoea since they can survive the often harsh conditions of the gut. In Bifidobacterium longum subsp. longum(T) NCIMB 702259, two gene clusters, with homology to the ATP-binding cassette (ABC) family of efflux transporters, were identified and studied to assess their functional contribution to antibiotic resistance. Both gene clusters contained two genes encoding putative efflux transporters and a regulator gene, upstream of the structural genes. Reverse transcriptase analysis indicated that the genes in each cluster were transcribed as operons, one where all three genes, including a putative MarR-type regulator were transcribed together (BLLJ_1496/1495/1494), and the other where the two ABC-type transporter genes (BLLJ_1837/1836) were co-transcribed, but excluded the putative regulator (BLLJ_1838). Heterologous expression of the cloned BLLJ_1837/1836 transporter genes in Lactococcus lactis conferred resistance to erythromycin and tetracycline by increasing the minimum inhibitory concentration between 1.5 and 3 fold. The presence of these genes also allowed a 16% increase in the efflux of Hoechst 33342 from L. lactis cells containing the two transporter genes, BLLJ_1837-6. In B. longum, an increase in the levels of transcription of 3.3 fold was observed for BLLJ_1837 in the presence of erythromycin, as measured by multiplex quantitative PCR. In contrast to this, the expression of the genes of the BLLJ_1495/1494 operon in L. lactis did not show significant drug resistance functionality. Gel shift experiments showed that in the BLLJ_1495/1494 operon, the putative MarR-type regulator protein (BLLJ_1496) bound with high affinity to the DNA sequence upstream of the operon in which it was located but this was not erythromycin dependent. This study demonstrated the occurrence of a drug inducible, ABC-type transporter system (BLLJ_1837/1836) in B. longum as well as a putative MarR-type DNA binding protein (BLLJ_1496).
施用益生菌，如双歧杆菌属，可以预防抗生素相关性腹泻，因为它们能够在肠道通常的恶劣条件下存活。在长双歧杆菌长亚种（模式菌株）NCIMB 702259中，鉴定并研究了两个与ATP结合盒（ABC）家族外排转运蛋白具有同源性的基因簇，以评估它们对抗生素抗性的功能贡献。两个基因簇均包含两个编码推定外排转运蛋白的基因和一个位于结构基因上游的调节基因。逆转录酶分析表明，每个簇中的基因作为操纵子转录，一个操纵子中所有三个基因，包括一个推定的MarR型调节基因一起转录（BLLJ_1496/1495/1494），另一个操纵子中两个ABC型转运蛋白基因（BLLJ_1837/1836）共同转录，但不包括推定的调节基因（BLLJ_1838）。将克隆的BLLJ_1837/1836转运蛋白基因在乳酸乳球菌中进行异源表达，通过将最低抑菌浓度提高1.5至3倍，赋予了对红霉素和四环素的抗性。这些基因的存在还使含有两个转运蛋白基因BLLJ_1837-6的乳酸乳球菌细胞中Hoechst 33342的外排增加了16%。在长双歧杆菌中，通过多重定量PCR测定，在存在红霉素的情况下，观察到BLLJ_1837的转录水平增加了3.3倍。与此相反，BLLJ_1495/1494操纵子基因在乳酸乳球菌中的表达未显示出显著的耐药功能。凝胶迁移实验表明，在BLLJ_1495/1494操纵子中，推定的MarR型调节蛋白（BLLJ_1496）与其所在操纵子上游的DNA序列具有高亲和力结合，但这并不依赖于红霉素。这项研究证明了长双歧杆菌中存在一种药物诱导型ABC型转运系统（BLLJ_1837/1836）以及一种推定的MarR型DNA结合蛋白（BLLJ_1496）。
