Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/12871
標題: 台灣西部地區豬隻與人類乳酸桿菌抗速博新與四環黴素之研究
Characterization of tetracycline and ciprofloxacin resistance lactobacilli isolated from swine and human at western area of Taiwan
作者: 張益銍
Chang, Yi-Chih
關鍵字: swine
豬隻
indigenous children
lactobacilli
ciprofloxacin
tetracycline
原住民小孩
乳酸桿菌
速博新抗生素
四環黴素
出版社: 獸醫學系暨研究所
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摘要: 乳酸桿菌是存在於人類及動物腸胃道中的一種正常菌叢。長久以來,腸道乳酸桿菌的數量一直被建議為動物健康狀況的指標,其部份菌株甚至被選為益生菌使用。乳酸桿菌對人類及動物的益處,已在增強免疫能力、促進動物生長、降低過敏現象、預防腹瀉以及對抗病原體等方面,有著廣泛的記載及證實。但是在過去的數十年裡,由於臨床醫學及農牧畜產業的廣泛濫用四環黴素和速博新抗生素,已造成一些腸道共生性細菌產生了此兩種藥物的抗藥性,並衍伸出許多重要的公共衛生問題。這些含有抗藥基因的細菌,在複雜的腸道微生物環境中,不但可成為抗藥基因的儲存槽,且常可藉由水平傳播,再將抗藥基因傳給其他病原性或伺機性細菌。 為瞭解台灣共生菌的抗藥情形,本研究首先分析台灣西部豬隻腸道中,乳酸桿菌的四環黴素抗性狀況。計由256隻豬的大腸檢體分離出159株抗四環素乳酸桿菌。經PCR及16S rRNA gene定序比對,確認了11種菌種 ( species ) 乳酸桿菌,包括:Lactobacillus reuteri, Lactobacillus amylovorus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus ruminis, Lactobacillus kefiri, Lactobacillus fermentum, Lactobacillus sakei, Lactobacillus coryniformis, Lactobacillus parabuchneri以及Lactobacillus letivazi。經抗生素敏感試驗分析,發現分離株有很高的MIC50及MIC90,顯示本省豬隻腸道中的乳酸桿菌具有高抗四環黴素的能力。接著以PCR的方法由菌種中偵測出5種抗四環黴素基因 ( tet genes )。其中以tet (W)基因的檢出率最高,接下來依序為tet (M), (L), (K)及(Q);它們的檢出率分别為82.0%,22.5 %,14.4 %,8.1%和0.9 %。值得注意的是,在相同菌種的分離株中,所帶的抗四環黴素基因種類雖相同,但抗藥的MIC值卻彼此常有很大的差異。此外,利用REP – PCR方法,來進行這些分離株的菌種亞型分析,發現這些分離株有著顯著的生物多樣性表現 ( biodiversity, % ),達62 %的平均值。 接著本研究分析台灣中部原住民小孩糞便中,乳酸桿菌對四環黴素與速博新抗生素之抗藥情形。結果由160個原住民小孩的糞便檢體,經選擇性培養分離及PCR–16S rRNA gene序列比對,鑑定出96株乳酸桿菌。其分別屬於6個菌種 ( species ) :L. plantarum, L. helveticusi, L. salivarius, L. casei, L. fermentum及L. pentosus。而在菌種亞型分析試驗中,以REP – PCR方法得知,這些分離株有著65.6 %的顯著生物多樣性。於藥物的最小抑菌濃度 ( MIC ) 分析裡,發現分離株對於速博新抗生素和四環黴素都有著很高且差異性很大的抗性,其對抗速博新抗生素的MIC50和MIC90都是64 ug/ml,而對抗四環黴素的MIC50和MIC90則分别為128 ug/ml和512 ug/ml。顯示台灣中部山區原住民兒童的腸道乳酸桿菌,已對速博新抗生素和四環黴素普遍有著高程度的抗性。
Lactobacilli are members of the normal microflora existing in the gastrointestinal ( GI ) tract of human and animals. For a long history, the lactobacilli level in intestine was suggested to be an index of animals' healthy status, from which isolates were generally regarded as safe and usually used as probiotics to promote the health of animals. The most widely documented effects of lactobacilli in humans and animals: improvement of weight and size, promotion of the immune system, reduction of alleragy, prevention of diarrhea, and exclusion of some pathogens. The emergence of tetracycline and ciprofloxacin resistance ( Tet-R and CIP-R ) in this commensal bacteria, after several decades' indiscriminate use of antibiotics in the fields of medicine and livestock breeding, has created an important public health problem, arousing much attentions in recent years, that this bacteria may act as reservoirs of antibiotic resistance ( AR ) genes and also as vehicles to horizontally transfer AR to opportunistic pathogens within complex microbial communities such as the gut microflora. To investipate the tetracycline resistance ( Tet-R ) condition of lactobacilli in Taiwan, we analyzed 256 pig colons and found that every sample contain typical colonies of Tet-R lactic acid bacteria. From these samples, a total of 159 isolates of Tet-R lactobacilli were obtained and identified as belonging to 11 species, including Lactobacillus reuteri, Lactobacillus amylovorus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus ruminis, Lactobacillus kefiri, Lactobacillus fermentum, Lactobacillus sakei, Lactobacillus coryniformis, Lactobacillus parabuchneri and Lactobacillus letivazi. All isolates had the significant high Tet-R MIC50 and MIC90 values, indicating an ecological distribution of Tet-R lactobacilli mostly with high resistance potency in pig colons. PCR-detection identified 5 tet genes in these isolates, the most predominant one being tet (W), followed by tet (M), (L), (K), and (Q). Their detection rates were 82.0 %, 22.5 %, 14.4 %, 8.1 % and 0.9 %, respectively. Noteworthily, isolates of the same species carrying identical tet gene(s) usually had a wide different MIC values. Furthermore, strain-subtyping of these isolates by REP-PCR demonstrated a notable genotypic biodiversity % ( average 62% ). Consecutively, we investigated the frequency of ciprofloxacin and tetracycline resistance lactobacilli in feces of aboriginal children. After an isolation on lactobacilli-selective medium and the subsequent analysis by the PCR-16S rRNA gene sequencing, a total of 96 isolates of lactobacilli were obtained and identified as belonging to 6 species, including L. plantarum, L. helveticusi, L. salivarius, L. casei, L. fermentum and L. pentosus. Strain-subtyping of these isolates by repetitive extragenic palindromic ( REP )-PCR demonstrated a notable genotypic biodiversity of 65.6 %. Antimicrobial susceptibility of ciprofloxacin and tetracycline had a wide different minimum inhibitory concentration ( MIC ) values in these isolates. The MIC50 and MIC90 of ciprofloxacin both was 64 ug/ml for both, while the MIC50 and MIC90 of tetracycline were 128 ug/ml and 512 ug/ml. These results indicate that high-level resistant activity of ciprofloxacin and tetracycline among Lactobacillus species in indigenous children's intestines were prevalent in mountain district at the central area of Taiwan.
URI: http://hdl.handle.net/11455/12871
其他識別: U0005-1101201219031100
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1101201219031100
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