探討克雷伯氏肺炎桿菌的colistin抗藥機轉

Investigation of colistin resistance mechanisms in Klebsiella pneumoniae isolates

洪贈薘(Zeng-Da Hong)

指導教授 ： 曾嵩斌

高雄醫學大學/健康科學院/醫學檢驗生物技術學系碩士班/碩士(2017年)

摘要

克雷伯氏肺炎桿菌是屬於革蘭氏陰性的腸內菌，除了對免疫力低下的人造成感染之外，也和許多院內感染有關。克雷伯氏肺炎桿菌在正常人呼吸道與糞便中也會發現此細菌，但對免疫力低下的病患則會造成許多不同的臨床症狀，如引起肺炎、腦膜炎、敗血症、肝膿瘍、泌尿系統感染等，若治療不當死亡率極高。Colistin又稱為polymyxin E，屬於多粘菌素 （polymyxin）類抗生素的其中一種，首次於1949年在日本由Bacillus polymyxa var. colistinus菌所分離出來，作用在革蘭氏陰性細菌細胞膜上特有的脂多醣 (Lipopolysaccharide)進而破壞細菌細胞膜的結構。目前colistin是院內感染中治療革蘭氏陰性細菌的後線用藥之一，但臨床分離的colistin抗藥性細菌比例卻逐漸上升值得注意。本研究主要探討colistin抗藥性的克雷伯氏肺炎桿菌之抗藥機轉，採用全台灣醫院所收集到對colistin呈現抗藥性的49株克雷伯氏肺炎桿菌臨床菌株，藉由PCR定序分析目前已知colistin的抗藥相關基因pmrA、pmrB 、crrB、phoP、phoQ、mgrB。在49隻菌株中，pmrA只有一株菌帶有有意義的胺基酸突變， pmrB有8株、crrB有4株、phoQ有6株、mgrB有3株、 pmrAB有3株、pmrB+crrB有1株、pmrB+phoP有1株、而pmrB+crrB+phoP+phoQ+mgrB有3株；此外，有20株以南方墨點法確認不具有crrB基因。為驗證胺基酸突變對colistin藥物感受性的影響，挑選數株進行互補實驗，將crrAB 基因片段clone至pOK12形成pOKcrrAB，以電穿孔儀將其送入待測菌株，檢測菌株的最低抑菌濃度 (minimum inhibitory concentration; MIC)變化：編號1610菌株帶有crrB (Q287K)突變，送入pOKcrrAB後colistin MIC由256 µg/ml下降至0.5 µg/ml；而編號723菌株帶有 crrB (L94M) 突變，在送入pOKcrrAB後colistin MIC由>1024 µg/ml下降至8 µg/ml。另挑選3株colistin MIC >1024µg/ml的菌株進行microarray分析，找尋其他有意義之抗藥相關基因，目前初步結果顯示colistin的抗藥機轉應為細胞膜上的LPS加上了帶正電的修飾，進而降低colistin的親和力導致抗藥性的產生。

關鍵字

克雷伯氏肺炎桿菌 ； 克痢黴素

並列摘要

Klebsiella pneumoniae is a Gram-negative pathogen often associated with nosocomial infections, including pneumonia, bacteremia, and urinary tract infection. The recent worldwide emergence of carbapenemase-producing K. pneumoniae (CRKP) isolates has been resulted in very limited therapeutic options. Colistin is one of the few remaining last-resort antibiotics that can be used to treat CRKP infection. Colistin are selectively toxic for Gram-negative bacteria due to their specificity for the lipopolysaccharide (LPS) molecule that exists within many Gram-negative outer membranes. However, resistance to colistin is increasingly reported from clinical settings. The colistin resistance mechanisms were discovered in 49 colistin-resistance K. pneumoniae isolates which were collected in Taiwan. The MICs (minimum inhibitory concentration) of colistin were determined by agar dilution according to the CLSI procedures. Colistin resistance genes (pmrA, pmrB, crrA, crrB, phoP, phoQ and mgrB) were detected and sequenced. Genetic mutations in clinical strains were including pmrA (n=1), pmrB (n=8), crrB (n=5), phoQ (n=6), mgrB (n=3), pmrAB (n=4), pmrB and crrB (n=1), pmrB and phoP (n=1), and pmrB, crrB, phoPQ and mgrB (n=3). In addition, Southern blotting confirmed that 20 CRKP isolates were without crrB. To determine whether the mutation in crrB was contributing to colistin resistance, complementation assay was performed. The crrAB of wild type gene was cloned to pOK12 and electroporated into the test strain. The variation of colistin MIC were tested in these strains. The colistin MIC of no. 1545 with pmrA (S64T), pmrB (P221S) and ccrB-deficient strains decreased from 8 µg/ml to 0.5 µg/ml. The colistin MIC of no. 1610 strain with crrB (Q287K) decreased from 256 µg/ml to 0.5 µg/ml. No.723 strain with crrB (L94M) decreased its MIC from > 1024 µg/ml to 8 µg/ml. Three strains of MIC > 1024 μg/ml were selected for microarray analysis. Two strains (No.44 and No.723) which had crrB mutation lead crrC expression, thereby upregulation of pmrHFIJKLM operon and pmrC (an effect mediated through the PmrAB two-component system) whereas the other strain (No.1721) which had pmrA mutant only lead upregulation of pmrHFIJKLM operon. Looking for other meaningful genes related to resistance, the current preliminary results show that colistin-resistant machine to the cell membrane of the LPS modification, thereby reducing the affinity of colistin lead to drug resistance.

並列關鍵字

Klebsiella pneumoniae ； colistin

參考文獻

Amako, K., Y. Meno, and A. Takade, Fine structures of the capsules of Klebsiella pneumoniae and Escherichia coli K1. J Bacteriol, 1988. 170(10): p. 4960-2.

2. Siu, L.K., et al., Klebsiella pneumoniae liver abscess: a new invasive syndrome. Lancet Infect Dis, 2012. 12(11): p. 881-7.

3. Hussein, K., et al., Carbapenem resistance among Klebsiella pneumoniae isolates: risk factors, molecular characteristics, and susceptibility patterns. Infect Control Hosp Epidemiol, 2009. 30(7): p. 666-71.

4. Gupta, N., et al., Carbapenem-resistant Enterobacteriaceae: epidemiology and prevention. Clinical infectious diseases, 2011. 53(1): p. 60-67.

5. Talbot, G.H., et al., Bad bugs need drugs: an update on the development pipeline from the Antimicrobial Availability Task Force of the Infectious Diseases Society of America. Clinical Infectious Diseases, 2006.