基因毒性克雷伯氏肺炎桿菌與大腸直腸癌之相關性:前驅性研究

Abstract

背景：在臺灣地區大腸直腸癌發生、死亡人數，每年呈快速增加的趨勢，根據台灣癌症登記中心的統計資料，在1979年到2015年近30年間大腸直腸癌的發生率增加了大約5倍，目前居所有癌症發生率及死亡率的第一位及第三位1。已有許多研究指出腸道微生物菌叢所製造的毒素可能是大腸直腸癌的危險因子之一，本研究欲探討的pks基因最早是在腸道外致病性的大腸桿菌中被發現，pks基因負責調控基因毒素Colibactin的合成，先前在細胞以及動物實驗證實帶有pks基因的克雷伯氏肺炎桿菌會造成DNA雙股螺旋斷裂，進而誘發大腸直腸癌，顯示帶有pks基因的克雷伯氏肺炎桿菌可能是大腸直腸癌的危險因子之一。另外也有流行病學研究發現，克雷伯氏肺炎桿菌肝膿瘍患者後續罹患大腸直腸癌的風險比起非克雷伯氏肺炎桿菌肝膿瘍患者來得高，顯示克雷伯氏肺炎桿菌與大腸直腸癌具有相關性，但目前流行病學的研究無法直接驗證帶有pks基因之克雷伯氏肺炎桿菌與大腸直腸癌的相關性。 研究目的：初步探討感染帶有pks基因的克雷伯氏肺炎桿菌是否會增加罹患大腸直腸癌的風險。 方法：本研究以常規收集臨床檢體之克雷伯氏肺炎桿菌菌株資料庫作為研究族群，首先挑選177株分離自肝膿瘍病人之克雷伯氏肺炎桿菌菌株，並利用PCR偵測pks基因上之clbA, clbB, clbN, clbQ基因座，確認菌株是否帶有pks基因，了解pks基因在高毒性克雷伯氏肺炎桿菌菌株中之分布狀況。另外藉由勾稽台大醫院癌症登記資料庫以及衛生福利部死因統計檔，確認病患之罹癌及存活情況，建立克雷伯氏肺炎桿菌菌株cohort，並計算cohort之大腸直腸癌標準化發生率比(standardized incidence rate ratio, SIR)，以及以pks盛行率隨機抽樣模擬計算帶有pks基因之克雷伯氏肺炎桿菌之大腸直腸癌標準化發生率比，初步分析感染帶有pks基因的克雷伯氏肺炎桿菌是否會增加罹患大腸直腸癌的風險。另外透過病例對照研究設計，了解pks基因在大腸直腸癌患者以及對照組間的分布是否有差異。 結果：分析177株肝膿瘍病患所分離出之克雷伯氏肺炎桿菌菌株，pks基因之盛行率為57.6%，高於一般臨床檢體所分離出來的克雷伯氏肺炎桿菌菌株之盛行率，其中pks基因主要分布於高毒性莢膜血清型K1 (78.8%)、K2 (41.7%)以及K20 (57.1%)之菌株中。全部克雷伯氏肺炎桿菌cohort之大腸直腸癌標準化發生率為1.3 (95%信賴區間 0.96- 1.69)。進一步區分，pks-positive克雷伯氏肺炎桿菌cohort大腸直腸癌標準化發生率比為2.0 (95%信賴區間1.19- 2.92)，達統計顯著；而pks-negative克雷伯氏肺炎桿菌cohort大腸直腸癌標準化發生率則為1.1 (95%信賴區間 0.71- 1.52)，未達統計顯著。病例對照研究發現大腸直腸癌患者所分離出之克雷伯氏肺炎桿菌株較一般來源族群分離出之克雷伯氏肺炎桿菌菌株有顯著較高的比例帶有pks基因 (30% vs. 16.7%, p<0.001)。 結論：本研究為第一個探討帶有pks基因的克雷伯氏肺炎桿菌與大腸直腸癌相關性之流行病學研究，我們的分析結果顯示克雷伯氏肺炎桿菌肝膿瘍患者中分離出之高毒性克雷伯氏肺炎桿菌菌株有將近60%帶有pks基因。標準化發生率比以及病例對照研究結果都支持pks-positive克雷伯氏肺炎桿菌與大腸直腸癌的相關性，因此未來值得繼續投入於這個重要的公共衛生議題。

Background: Colorectal cancer (CRC) is the most common cancer and the third leading cause of cancer-related death in Taiwan. The risk factors for CRC include genetic factors, life style factors, and microbial etiology such as colibactin-producing intestinal bacteria. Recently, studies found that Klebsiella pneumoniae with pks colibactin gene may induce carcinogenesis in vivo and in vitro. The higher rate of CRC in patients with K. pneumoniae pyogenic liver abscess (PLA) than in those with non-K. pneumoniae PLA also supported that K. pneumoniae might be the microbial etiology of CRC. Therefore, we used a case-control study and standardized incidence rate ratio (SIR) calculation to preliminary investigate the link between pks-positive K. pneumoniae and colorectal cancer. Aims: To preliminary investigate the link between pks-positive K. pneumoniae and colorectal cancer. Material: Our K. pneumoniae cohort is comprised of 8,675 K. pneumoniae strains from patients at National Taiwan University Hospital and Yunlin branch from 2004 to 2018. The presence of pks colibactin genes among K. pneumoniae strains were determined by PCR with primer for clbA, clbB, clbN, and clbQ on the pks gene cluster. We will determine whether and when the cohort subjects were diagnosis to had colorectal cancer by crosslinking Taiwan cancer registry. We will use the colorectal cancer incidence of age-, sex- and year-matched Taiwan general population as the external comparison group to calculate the standardized incidence rate ratio (SIR). The SIR is defined as the ratio between numbers of observed cases O and numbers of expected cases E. E is calculated by multiplying the number of person-years by the colorectal cancer incidence rates for each 5-year age groups, and 5-year calendar periods of the cohort members, the 95% confidence intervals (CIs) are based on the Poisson distribution. We also conduct a case-control study to determine whether the distribution of pks gene in case and control group different. Results: The prevalence of pks gene was 57.6% in the K. pneumoniae strains isolated from pyogenic liver abscess patients, and predominately in K1 (78.8%), K2 (41.7%) and K20 (57.1%) hypervirulence capsular type. The CRC standardized incidence rate ratio (SIR) of the whole KP cohort was 1.3 (95% CI 0.96- 1.69). The result of pks-positive SIR was 2.0 (95%CI 1.19- 2.92) and pks-negative SIR was 1.1 (95%CI 0.71- 1.52). In case-control study, the results show that the rate of pks gene was significantly higher in the K. pneumoniae strains isolated from CRC patients and the pks rate in case and control were 30% vs. 16.7% (p<0.001). Conclusions: Our results of SIR and pilot case-control study preliminarily support our hypothesis that the carriage of pks gene cluster may serve as a molecular basis underlying the epidemiological link between K. pneumoniae and CRC.