抗生素引起之共生菌相失衡促使超級細菌在小鼠腸道定殖和入侵

Abstract

對beta-內酰胺有抗藥性的超級細菌，例如擁有新德里金屬beta-內酰胺酶(NDM)-1的超級細菌，對公眾健康有極大的威脅。腸道內的共生菌叢促進腸道屏障功能的穩固，並且與外來病菌競爭。而目前已知道抗生素會擾亂腸道共生菌叢。 目的：了解由抗生素造成的腸道菌叢擾亂，是否會促進超級細菌在腸道內生長和轉移。 方法： BALB/c小鼠給予正常飲水或是抗生素飲水七天，抗生素水去除之後，每隻小鼠灌食109CFU抗氨芐青黴素的大腸桿菌進行感染，在感染後的第0, 1, 3, 7, 和14天進行犧牲。利用蘇木紫-伊紅染色分析腸道的結構，並計數在腸道、脾臟、肝臟的細菌數量。被腸道上皮細胞內吞的細菌數量，則由慶大黴素耐藥檢測分離出來。利用螢光原位雜交，了解細菌入侵到黏膜層的情形。結果： 在第0天，抗生素組的腸道菌叢數量比正常飲水組的低。抗氨芐青黴素的細菌在正常飲水組的腸道中，每個時間點都沒有生長的現象。相反地，抗生素組的小鼠腸道中，在感染後第1, 3天都有抗氨芐青黴素的細菌出現在空腸、盲腸和結腸。抗氨芐青黴素的大腸桿菌在第7和14天被從腸道清除。此外，抗生素組小鼠在感染後第1天，出現盲腸腫脹並且有組織充血及白血球浸潤造成的水腫現象。另外，感染後第三天有細菌入侵空腸腺窩，空腸和結腸也有細菌被腸道上皮細胞內吞的情形。感染後第1和3天，有細菌在脾臟和肝臟轉移的現象。 結論： 正常的腸道菌叢具有屏障功能，可保護腸道不受抗藥性細菌感染。腸道菌叢受到干擾則會促進抗藥性細菌之定殖，並造成腸道共生菌和超級細菌都會體內的散布。

Superbugs that are resistant to beta-lactams antibiotics, such as those with New Delhi metallo-beta-lactamase (NDM)-1, pose major threats to public health. Enteric commensal microflora is involved in mucosal barrier fortification and pathogen competition. Antibiotics are known to disrupt intestinal flora. Aim: The aim is to evaluate whether antibiotic-induced intestinal dysbiosis may promote enteric colonization and translocation of superbug. Methods: BALB/c mice were drinking normal water (NW) or antibiotic water (AW) for 7 days. Ampicillin-resistant (Amp-r) E. coli BL21 (109 CFU) was administered by oral gavage after antibiotic withdrawal. Animals were sacrificed at 0, 1, 3, 7 and 14 days after inoculation. The structure of intestine was determined by H&E staining. Bacterial colony forming units in intestine, liver and spleen were assessed. The amount of intracellular bacteria in purified enterocytes was determined using a gentamicin resistance assay. Bacterial invasion to mucosa was determined by fluorescent in situ hybridization. Results: The enteric bacterial counts were reduced in AW mice compared to NW groups on day 0. After inoculation of Amp-r E.coli, no sign of bacterial colonization and translocation was seen in NW mice throughout all time points. In contrast, AW mice showed Amp-r E. coli in the jejunum, cecum and colon after inoculation for 1 and 3 days. Clearance of Amp-r E. coli was associated with recovery of commensal bacterial numbers after 7 and 14 days. Moreover, cecal flatulence and tissue edema associated with hyperemia and leukocyte infiltraton were observed in AW mice on day 1 post-infection. Furthermore, bacterial invasion to jejunal crypts, bacterial endocytosis in jejunal and colonic enterocytes, and bacterial translocation to liver and spleen were observed on day 1 and 3 post-infection in AW mice. Conclusions: The normal commensals served as a barrier to protect the intestine from antibiotic-resistant bacterial colonization. Enteric dysbiosis predisposes antibiotic-resistant bacteria to colonize, leading to systemic dissemination of both commensals and superbug.