克雷白氏肺炎菌對imipenem之抗藥機轉研究

Abstract

多重抗藥性革蘭氏陰性菌是院內感染及管控問題中的燙手山芋，由於造成治療上極大的困擾，影響病患存活率及院內醫療費用。近年來許多國家陸續發現對carbapenem具抗藥性K.pneumoniae院內感染群突發，因此本篇研究發展有效可行的抗菌策略亦瞭解該菌的致病機轉。研究63株克雷白氏肺炎菌使用抗生素imipenem的最低抑菌濃度( Minimum inhibitory concentration; MIC )，鑑定以藥敏試驗賀治試驗(Modified Hodge test)，確定其菌對該抗生素具有carbapenemase。以PCR方法確認carbapenem抗藥性基因IMP, VIM, GIM, GES, OXA, KPC, NDM篩檢含β-lactamase腸內菌。六十三株菌中有19株對於賀治試驗結果顯示陽性。根據PCR結果指出14.3%所研究的臨床菌株繫帶bla-IMP，另外2.38%並含bla-VIM、KPC抗藥基因。為了深入探討細菌的抗藥機轉，以PCR及賀治試驗篩選過後的菌株當基礎，建構抗carbapenem菌株的表現基因庫，來分析對imipenem具抗藥性的重組基因。從臨床菌株9921及7285中，與負控制菌ATCC 25922比較，前者共有十三株重组殖株(recombinant clones)的MIC提升四倍，而後者共有七株殖株提升兩倍的MIC。本研究利用表現基因庫及基因比對選殖克雷白氏肺炎菌之多重藥物抗藥性相關基因，經由PCR及DNA定序測試此表現基因庫之代表性。之後以1 μg/ml imipenem及50 μg/ml kanamycin之LB 固態培養基篩選出對imipenem具抗藥性之XLOLR菌株，並抽取出其內部帶有K.pneumoniae基因片段之質體，經由再次轉型作用 (transformation) 送入另一株SOLR菌株，最後驗證再次轉型菌株對imipenem 最小抑菌濃度，以確認所挑選出的MDRKP基因確實與imipenem抗藥性有關。研究評量NTUH-9921和7285基因庫包含範圍為90.9%及95.8%，分析發現NTUH-9921菌株之基因庫有表現ESBL和Amp-C酶，而Amp-C酶中大多類是DHA及CTX-M型。此外，7285菌株之基因庫則表現鐵離子嵌合物(siderophores)、Amp-C酶，及外排泵基因 (efflux genes)。

Multidrug resistant gram negative organisms have become an increasingly difficult problem in hospitals, due to its impact on length of hospital stay, morbidity, mortality, and increasing the cost of care. Beta-lactamases are the most common resistance modality in Gram-negative pathogens, where carbapenem antibiotics, such as imipenem, have been drug treatment for serious infections caused by these pathogens. Resistance to carbapenems has been uncommon until now. Recently, Klebsiella pneumoniae has been found possessing resistance to carbapenems, Therefore in this study it is our interest to study the prevalence of acquisation of common carbapenemase genes as well as the specific resistance mechanism found in Taiwanese clinical strains. In a set of 62 K. pneumoniae and a K. oxytoca isolates acquiring imipenem resistance as seen from minimum inhibitory concentration examinations; carbapenemase was suggested on the basis of Modified Hodge Test and was then confirmed by PCR in order to screen out isolates possessing common carbapenemase genes. PCR was performed to screen for IMP, VIM, GIM, GES, OXA, KPC, and NDM. For the Modified Hodge Test, 27 out of 62 tested isolated strains tested positive. For PCR, 14.3% of our clinical isolates were IMP-positive, 2.38% were VIM-positive, and 2.38% were KPC-positive. As for the rest of the isolates in which Modified Hodge Test appeared prominent, while PCR demonstrated negative results, expression libraries were constructed for strains that were highly resistant. We hypothesize unknown mechanisms of imipenem resistance within our bacterial strains. By using the ZAP express cloning approach, we generated library of clones for NTUH resistant strains 9921 and 7285. Library coverage of 90.9% for 9921 strain and 95.8% for the 7285 strain was estimated. As compared to our negative control ATCC 25922 E.coli, the minimum inhibitory concentration of thirteen clones from resistant strain 9921 genetic library screened to increase four fold, while 7285’s genetic library revealed seven clones with two fold increasement. After a complete construction of both genetic libraries, our aim of expression cloning identified specific enzymes aiding imipenem resistance in Klebsiella pneumoniae. It has been found that 9921 resistance is due to the expression of AmpC and CTX-M enzyme. On the other hand, 7285 strain expressed siderophore-binding proteins, AmpC enzyme as well as the multidrug efflux system MdtA. Two clones expressing AmpC and CTX-M carbapenemase enzymes from 9921’s library were selected, and its plasmid retransformed into imipenem-susceptible XLOLR strain. The minimum inhibitory concentration to imipenem increased four fold after retransformation Due to the increase in minimum inhibitory concentration; a mutant library was constructed for 7285 resistant strain in order to analyze the correlation of our mutant clones to the resistance of imipenem. This study shows an increase of imipenem-nonsusceptible K.pneumoniae in Taiwan, due to impact of multiple resistance mechanisms.