探討造成社區性感染及院內感染之克雷伯氏肺炎桿菌對氨芐青黴素抗藥性之機制

Abstract

克雷伯氏肺炎桿菌（Klebsiella pneumoniae）是現今常見於院內感染及社區型感染的病原菌，其抗藥性發展日益嚴重，不只已經對廣譜的乙內醯胺（β-lactam）類抗生素產生大幅的抗藥性，連原本能有效治療具有廣效性乙內醯胺酶（Extended spectrum β-lactamases）之克雷伯氏肺炎桿菌感染的碳青黴烯類（Carbapenem）抗生素也失去功效。最早被發現克雷伯氏肺炎桿菌具有抗藥性的抗生素是隸屬於青黴素類 （Penicillin）的氨芐青黴素（Ampicillin），起因於克雷伯氏肺炎桿菌染色體上本來就有編碼盤尼西林酶（Penicillinase）的基因shv-1，因此能水解這類抗生素。本研究想了解在台大醫院收集到之臨床菌株NTUH-K2044對於氨芐青黴素具有抗藥性，其機制為何。因此透過先前實驗室已建立好帶有mini-Tn5跳躍基因的NTUH-K2044菌株跳躍子突變株庫（Transposon mutant library）來篩選目標突變株，篩到了兩株突變株在100 μg/mL的氨芐青黴素下生長情況相對不佳，進而定序比對結果。發現跳躍子插入guaB基因中，此基因的產物為肌苷單磷酸去氫酶（Inosine 5'-monophosphate dehydrogenase，IMPDH），在三磷酸鳥苷（Guanosine triphosphate，GTP）和鳥嘌呤（Guanine）的合成途徑上是重要的催化酵素，也會進而影響DNA和RNA合成以及細胞增生。因此，我們將此基因剔除，觀察其最小抑菌濃度（Minimum inhibitory concentration，MIC）變化。接著對剔除guaB基因後的菌株做了鳥嘌呤回補試驗，發現無論氨芐青黴素加入與否，都不影響突變株，因此推論guaB基因影響NTUH-K2044生長，並非參與對氨芐青黴素的抗藥機制。之後利用NTUH-K2044已知的全基因序列（Whole genome sequence）進行比對，找出其基因體上和乙內醯胺酶相關的基因shv11，建構缺失突變株後測試其氨芐青黴素的最小抑菌濃度，比起參考菌株下降了32倍，表示shv11確實參與了NTUH-K2044對氨芐青黴素的抗藥機制。此外，也針對一株造成院內感染的碳青黴烯類耐藥克雷伯氏肺炎桿菌菌株（carbapenem-resistant Klebsiella pneumoniae，CRKP）CB28進行乙內醯胺酶相關基因的剔除，分別是shv1、shv2、toho1、kpc基因，已成功獲取shv1、toho1、kpc等三株缺失突變株及kpc、toho1雙重基因缺失突變株，但進行抗生素感受試驗時，其最小抑菌濃度皆沒有下降，直到使用舒巴坦（Sulbactam）乙內醯胺酶抑制劑輔助氨芐青黴素活性時shv1、kpc基因缺失突變株及kpc、toho1雙重基因缺失突變株才有下降大於4到8倍的現象。

Klebsiella pneumoniae is a common pathogen responsible for both hospital-acquired and community-acquired infections. The development of antimicrobial resistance in this bacterium has become a serious concern, as it has not only acquired extensive resistance to broad-spectrum β-lactam antibiotics but also rendered carbapenem, which were once effective against extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae infections. The initial discovery of antibiotic resistance in K. pneumoniae was associated with ampicillin, a member of the penicillin class, due to the presence of the gene shv1 encoding penicillinase on the bacterial chromosome, which could hydrolyze such antibiotics. This study aimed to understand the mechanisms of resistance to ampicillin in clinical strain NTUH-K2044 collected from National Taiwan University Hospital. To investigate this, a transposon mutant library of NTUH-K2044 containing mini-Tn5 transposons was employed for screening target mutants. Two mutants with compromised growth under 100 μg/mL of ampicillin were identified and subjected to sequencing analysis. The insertion of the transposon in the guaB gene was found, which encodes inosine 5'-monophosphate dehydrogenase (IMPDH), a critical catalytic enzyme involved in the synthesis of guanosine triphosphate (GTP) and guanine, affecting DNA and RNA synthesis as well as cell proliferation. However, the deletion of the guaB gene in NTUH-K2044 was performed, and the changes in the minimum inhibitory concentration (MIC) of ampicillin were observed. In the guanine supplementation test, the growth of the guaB mutant strains was not affected, regardless of the presence of ampicillin. Consequently, we hypothesize that the guaB gene affects the growth condition of NTUH-K2044 but does not participate in the resistance mechanism. Further analysis using the whole-genome sequence of NTUH-K2044 identified the presence of the β-lactamase-associated gene shv11. Constructing a shv11 deletion mutant and testing its MIC against ampicillin showed a 32-fold decrease compared to the reference strain, indicating the involvement of shv11 in ampicillin resistance in NTUH-K2044. Additionally, the study focused on a carbapenem-resistant Klebsiella pneumoniae (CRKP) strain, CB28, causing hospital-acquired infections. Deletion β-lactamase-associated genes, including shv1, shv2, toho1 and kpc were performed. Although three single gene deletion mutants （shv1, toho1 and kpc） and one double gene deletion mutant （kpc and toho1） were obtained, none of them showed reduced MIC against ampicillin. However, the combination of sulbactam, a β-lactamase inhibitor, and ampicillin led to a significant 4 to 8-fold decrease in MIC for shv1, kpc single gene deletion mutants and kpc. toho1 double gene deletion mutant.