克雷伯氏肺炎杆菌之比较基因体学研究

克雷伯氏肺炎桿菌之比較基因體學研究

Comparative Genomics Study of Klebsiella pneumoniae

吳克銘

Keh-Ming Wu

生物醫學資訊研究所

關鍵詞:

克雷伯氏肺炎桿菌;基因體定序;比較基因體學;蛋白質序列交互比較;基因體雜交技術;

Keyword:

Klebsiella pneumoniae;genome sequencing;comparative genomics;Reciprocal Protein Sequence

Comparison;CGH;

摘要:

克雷伯氏肺炎桿菌為一革蘭氏陰性腸內細菌，自1882年發現以來，至今仍是造成人類疾病的

重要病原菌。克雷伯氏肺炎菌透過院內感染以及社區感染所造成之臨床表現隨著不同年代和

地域而有所差異，除了肺炎與尿道感染為其傳統病徵之外，同時它也是造成菌血症與抗藥性

細菌感染之新興病源。近三十年來，在台灣以及鄰近亞洲各國，出現一種好發於糖尿病病患

的侵犯性克雷伯氏肺炎桿菌感染症，能引起原發性肝膿瘍合併菌血症、轉移性內眼炎或腦膜

炎的發生。雖然目前對於克雷伯氏肺炎桿菌在亞洲造成嚴重感染症之主要原因仍不清楚，但

推測可能與宿主及菌株因子兩者都有相關。 我們對於同一種細菌會因帶有不同基因內涵而

造成不同臨床症狀有興趣，因此我們採用散彈槍定序策略完成一個克雷伯氏肺炎桿菌致病性

菌株NTUH-K2044之全部基因體序列。此一菌株對人體能造成侵犯性感染並對小鼠具有致死性毒

性。與另一株取自肺炎病患並由華盛頓大學所定序的菌株MGH 78578做序列分析比較，發現六處

基因叢集有所不同，包含與檸檬酸發酵途徑、脂多醣合成、莢膜多醣合成、噬菌體、纖毛等

相關之基因叢集。運用本實驗室自行建立之Genomic Shotgun Array（GSA）技術，將所收集之15株由

社區感染或醫院感染而來的臨床菌株與NTUH-K2044菌株進行基因體雜交比較，我們可以區分出

三群細菌，其基因體內容型式與臨床病徵、抗藥性種類、小鼠模式所得之毒性特徵相符。

為了瞭解克雷伯氏肺炎桿菌感染之分子機制並發展有效之治療方法，我們進一步研究克雷

伯氏肺炎桿菌之基因體演化、致病性及抗藥性。為此，我們挑選三株 (NK8, NK29, NK245) 具有不

同抗藥性、臨床特徵與採菌歷史之菌株進行全基因體定序。如同預測，我們發現抗藥菌株帶

有多重抗藥性基因的質體：菌株NK245的一個質體pK245帶有qnrS 喹諾酮(quinolone)類抗藥基因

與SHV-2乙內醯胺酶基因(beta-lactamase)，而菌株NK29的一個質體pK29帶有CMY-8乙內醯胺酶基因與CTX-M-3 廣效性乙內醯胺酶(extended-spectrum beta-lactamase)基因。 為了快速鑑定克雷伯

氏肺炎桿菌的基因體內容，我們利用NimbleGen oligonucleotide array (NOA) 發展第二代基因體雜交技

術。微陣列上的探針是設計自五株 (NTUH-K2044, NK8, NK29, NK245 and MGH 78578)

已完成定序的克雷伯

氏肺炎桿菌的非重複編碼序列。將所收集之26株臨床菌株(包含GSA所用的14株)與參考菌株NTUH-K

2044進行基因體雜交比較並利用階層式分群法進行分析，結果顯示NOA技術不僅能將菌株基因

內涵型態與其表現型特徵作良好關聯，NOA技術還具有更高解析度、涵蓋更多基因內涵與降低

實驗所需之操作等優點。 使用本人所寫的「蛋白質序列交互比較程式」分析六株已完成

定序的克雷伯氏肺炎桿菌，發現不同菌株間相對應的基因在染色體上的排列相當一致。然而

在大腸桿菌即使在親緣關係非常接近的K12亞種與O157:H7亞種裡也能發現明顯的基因片段倒置

。此結果顯示克雷伯氏肺炎桿菌相較於其他腸內菌科的細菌，如大腸菌，擁有相對穩定的染

色體。 為了瞭解親緣關係相近菌種其基因在演化上的動態變化，分別計算克雷伯氏肺炎

桿菌與大腸桿菌各自核心基因群的Ka/Ks值，以及核心基因群依照功能分類後所各自計算的Ka/K

s值，結果都分別小於1，顯示克雷伯氏肺炎桿菌與大腸桿菌這兩個菌種都受到強烈的負向天

擇作用，使得有害的突變大多都被移除。然而，在依照功能分類後所各自計算的核心基因Ka/

Ks值卻在兩個菌種中不盡相同，顯示在克雷伯氏肺炎桿菌與大腸桿菌之不同功能的基因群承

受著不同的演化壓力。

Abstract:

Klebsiella pneumoniae is a Gram-negative enteric bacillus that has been a significant human pathogen

since its discovery in 1882. The clinical manifestations of K. pneumoniae infection acquired from hospital or community vary in different time periods and geographical areas. Besides its classic manifestation as

pneumonia and urinary tract infection, K. pneumoniae is emerging worldwide as a major cause of bacteremia and drug-resistant infection. Moreover, in the past three decades, an invasive form of K. pneumoniae infection, which presents as primary bacteremic liver abscesses, metastatic endophthalmitis or meningitis, has been reported to be associated with diabetes mellitus and occurred almost exclusively in Asia, including Taiwan. Although the reasons for the preponderance of this severe invasive K. pneumoniae infection in Asia are unknown, they are likely to involve both host and microbial factors. We are interested in understanding how differences in gene content of the same bacterial species result in variable diseases in human infection. Therefore, we have determined the whole genome sequence of K. pneumoniae NTUH-K2044, a virulent strain causing invasive phenotype in humans and lethality in mice, and compared

to that of strain MGH 78578, which was isolated from a patient with pneumonia in USA. Major genomic differences were found in six gene clusters that included an integrative and conjugative element, clusters involved in citrate fermentation, lipopolysaccharide synthesis, and capsular polysaccharide synthesis,

phage-related insertions, and a cluster containing fimbria-related genes. We also conducted comparative genomic hybridization on 15 K. pneumoniae isolates obtained from community-acquired or nosocomial infections using tiling probes for the NTUH-K2044 genome (GSA method). Hierarchical clustering revealed three major groups of genomic insertion-deletion patterns that correlated with the strainsÕ clinical features, antimicrobial susceptibilities, and virulence phenotypes with mice. To further elucidate the genomic contents of drug-resistant K. pneumoniae strains and gain knowledge about the evolution of enteric bacteria, three hospital isolates, NK8, NK29 and NK245, have been selected for whole genome shotgun sequencing based on their isolation history and antibiotics resistance profile. As expected, two plasmids harbor multiple antimicrobial resistance genes were found. The plasmid pK245 contains the qnrS quinolone resistance determinant and the gene encoding SHV-2 beta-lactamase in strain NK245, and the plasmid

pK29 contains genes encoding CMY-8 beta-lactamase and CTX-M-3 ESBL in strain NK29. To rapidly identify the patterns of K. pneumoniae gene contents, we have developed another CGH method using NimbleGen oligonucleotide array (NOA). The probes were designed based on all non-redundant coding sequences from five completed genomes (NTUH-K2044, NK8, NK29, NK245 and MGH 78578). Twenty-six strains (including 14 used in GSA) were analyzed by comparative genomic hybridization using the

NTUH-K2044 as the reference strain. The hierarchical clustering results indicate that the NOA method not only can correlate gene contents with phenotype features, but also provide the advantages of higher resolution, better gene-content coverage and less laboriousness. Using the self-developed ÒReciprocal Protein Sequence ComparisonÓ program, we found the coding genes are highly syntenic along the chromosomal coordinates among K. pneumoniae isolates. By contrast, within E. coli, inversions could be found between substrains of K12 and O157:H7. This finding suggests that K. pneumoniae has a relatively stable chromosome compared to E. coli and other species within Enterobacteriaceae. To understand the evolutionary dynamics of protein-coding sequences across closely related species, the Ka/Ks ratios were calculated for K. pneumoniae and E. coli core genes and analyzed for each COG functional groups. In either case we found an average Ka/Ks ratio of less than 1, suggesting both species were subjected to strong purifying selection to eliminate harmful mutations. However, some functional groups showed different

Ka/Ks ratios in K. pneumoniae and E. coli, indicating that genes of different function groups had subjected to different selection pressures for the two related enterobacteria during evolution.