Lipopolysaccharide O1 Antigen Contributes to the Virulence in Klebsiella pneumoniae Causing Pyogenic Liver Abscess

Klebsiella pneumoniae is the common cause of a global emerging infectious disease, community-acquired pyogenic liver abscess (PLA). Capsular polysaccharide (CPS) and lipopolysaccharide (LPS) are critical for this microorganism's ability to spread through the blood and to cause sepsis. While CPS type K1 is an important virulence factor in K. pneumoniae causing PLA, the role of LPS in PLA is not clear. Here, we characterize the role of LPS O antigen in the pathogenesis of K. pneumoniae causing PLA. NTUH-K2044 is a LPS O1 clinical strain; the presence of the O antigen was shown via the presence of 1,3-galactan in the LPS, and of sequences that align with the wb gene cluster, known to produce O-antigen. Serologic analysis of K. pneumoniae clinical isolates demonstrated that the O1 serotype was more prevalent in PLA strains than that in non-tissue-invasive strains (38/42 vs. 9/32, P<0.0001). O1 serotype isolates had a higher frequency of serum resistance, and mutation of the O1 antigen changed serum resistance in K. pneumoniae. A PLA-causing strain of CPS capsular type K2 and LPS serotype O1 (i.e., O1:K2 PLA strain) deleted for the O1 synthesizing genes was profoundly attenuated in virulence, as demonstrated in separate mouse models of septicemia and liver abscess. Immunization of mice with the K2044 magA-mutant (K1− O1) against LPS O1 provided protection against infection with an O1:K2 PLA strain, but not against infection with an O1:K1 PLA strain. Our findings indicate that the O1 antigen of PLA-associated K. pneumoniae contributes to virulence by conveying resistance to serum killing, promoting bacterial dissemination to and colonization of internal organs after the onset of bacteremia, and could be a useful vaccine candidate against infection by an O1:K2 PLA strain

Capsular Types of Klebsiella pneumoniae Revisited by wzc Sequencing

Capsule is an important virulence factor in bacteria. A total of 78 capsular types have been identified in Klebsiella pneumoniae. However, there are limitations in current typing methods. We report here the development of a new genotyping method based on amplification of the variable regions of the wzc gene. Fragments corresponding to the variable region of wzc were amplified and sequenced from 76 documented capsular types of reference or clinical strains. The remaining two capsular types (reference strains K15 and K50) lacked amplifiable wzc genes and were proven to be acapsular. Strains with the same capsular type exhibited &gt;= 94% DNA sequence identity across the variable region (CD1-VR2-CD2) of wzc. Strains with distinct K types exhibited &lt;80% DNA sequence identity across this region, with the exception of three pairs of strains: K22/K37, K9/K45, and K52/K79. Strains K22 and K37 shared identical capsular polysaccharide synthesis (cps) genes except for one gene with a difference at a single base which resulted in frameshift mutation. The wzc sequences of K9 and K45 exhibited high DNA sequence similarity but possessed different genes in their cps clusters. K52 and K79 exhibited 89% wzc DNA sequence identity but were readily distinguished from each other at the DNA level; in contrast, strains with the same capsular type as K52 exhibited 100% wzc sequence identity. A total of 29 strains from patients with bacteremia were typed by the wzc system. wzc DNA sequences confirmed the documented capsular type for twenty-eight of these clinical isolates; the remaining strain likely represents a new capsular type. Thus, the wzc genotyping system is a simple and useful method for capsular typing of K. pneumoniae

Isolation of genetic loci associated with phagocytosis and virulence in Klebsiella pneumoniae using a Dictyostelium model

吞噬作用是宿主對抗致病微生物的第一道防線，因此，對於防禦細菌入侵扮演重要的角色。而在細菌中同樣也有重要的致病基因負責抵禦吞噬作用，進而成功的感染人體。克雷伯氏肺炎桿菌(Klebsiella pneumoniae)引起之化膿性肝膿瘍為台灣重要之新興感染症，其致病機制目前所知和先天免疫中之血清與吞噬抗性最相關。本實驗室原來利用黏性去篩選一突變株庫，結果得到一致病基因為magA，後來證實是負責K1莢膜多醣體合成的重要基因。由於用黏性之篩檢侷限於莢膜與外壁相關的基因，為了能進一步篩選與吞噬抗性有關之基因，本研究使用黏菌(Dictyostelium discoideum)模式進行篩選。黏菌的變形蟲(amoeba)時期與哺乳類的巨噬細胞有許多相似的特徵，其一便是具有吞噬並殺死細菌的能力。本研究以此模式篩選造成肝膿瘍且對於吞噬作用具有抗性的克雷伯氏肺炎桿菌(NTUH-K2044)之突變株庫，找尋與抵抗吞噬作用相關的致病基因，約2500株突變株之中有29個突變株可產生溶菌圈。分析跳躍子嵌入之位置，共有六個基因，其中三個為負責莢膜合成之基因，另外三個分別為KP2125(肉鹼carnitine代謝相關)，clpX(蛋白酶ATPase分子)以及wzm基因(脂多醣O抗原之運輸蛋白)，再一次將基因剔除以及將此基因補回，並確認吞噬作用的結果發現只有有△clpX 以及△wzm 基因剔除株對黏菌吞噬失去抗性，而基因補回株也恢復其抗性。此外，由健康人周邊血液分離的噬中性白血球與菌株作用證實兩突變株的確較易被人類的噬中性白血球吞噬清除。之後藉由動物實驗發現兩突變株感染之小鼠相較於野生株感染的小鼠存活率高，顯示這兩個基因確實影響克雷伯氏肺炎桿菌的致病力。此外，同時發現與clpX屬於同一個操縱基因組(operon)的clpP 基因對於抗吞噬作用也是重要的。利用基因微陣列進一步分析△clpX 基因剔除株中基因表現情形發現其莢膜合成基因表現量相較於野生株下降了3 倍，但莢膜合成量減少並不是此突變株抵抗吞噬作用能力下降的唯一原因。因此，本研究利用黏菌模式發現對於克雷伯氏肺炎桿菌抗吞噬與致病重要的基因，也證實黏菌模式可用於具有厚層莢膜之細菌以進行與吞噬抗性相關基因的篩選。Phagocytosis is the first line of host defense against invading microorganisms, therefore, it plays an important role in host antibacterial response. On the other hand, some bacterial genes responsible for attacking phagocytosis may be essential for their pathogenicity. Klebsiella pneumoniae causing pyogenic liver abscess is an important emerging infectious disease in Taiwan. Resistance to innate immune response —serum killing and phagocytosis are known to be critical virulence factors in K. pneumoniae. Using transposon mutagenesis and mucoviscosity screening, we had identified a virulent gene, magA, related to phagocytic resistance. This gene was further proved to be associated with synthesis of K1 capsule. Because the limitation of screening by mucoviscosity, we use a Dictyostelium model to identify genes associated with phagocytic resistance. Since the amoeba form of Dictyostelium discoideum and mammalian macrophage share many traits such as the ability to phagocytose and kill bacteria, in this study, we used a Dictyostelium model to investigate genes for resistance to phagocytosis in NTUH-K2044, a strain of K. pneumoniae causing pyogenic liver abscess that is highly resistant to phagocytosis. A total of 2500 transposon mutants were screened by plaque assay, and 29 of them permitted phagocytosis by Dictyostelium. In the 29 mutants, 6 loci were identified; three were capsular synthesis genes. Among the other three, one was related to carnitine metabolism (KP2125), one encoded a subunit of protease (clpX), and the other encoded a lipopolysaccharide O-antigen transporter (wzm). Deletion and complementation of these genes showed that only △clpX and △wzm mutants became susceptible to Dictyostelium phagocytosis, and their complementation restored the phagocytosis resistance phenotype. These two mutants were also susceptible to phagocytosis by human neutrophils and revealed attenuated virulence in a mouse model, implying that they play important roles in the pathogenesis of K. pneumoniae. Furthermore, we demonstrated that clpP, which exists in an operon with clpX, was also involved in resistance to phagocytosis. The transcriptional profile of △clpX was examined by microarray and revealed a 3-fold lower level of expression of capsular synthesis genes. However, decrease of capsule synthesis was not the only reason for impaired phagocytosis resistance. Therefore, we have identified genes involved in resistance to phagocytosis and virulence in K. pneumoniae using Dictyostelium, and this model is useful to explore genes associated with resistance to phagocytosis in heavily-encapsulated bacteria

Genetic analysis of capsular polysaccharide synthesis gene clusters in 79 capsular types of Klebsiella spp

A total of 79 capsular types have been reported in Klebsiella spp., whereas capsular polysaccharide synthesis (cps) regions were available in only 22 types. Due to the limitations of serotyping, complete repertoire of cps will be helpful for capsular genotyping. We therefore resolved the rest 57 cps and conducted comparative analysis. Clustering results of 1,515 predicted proteins from cps loci categorized proteins which share similarity into homology groups (HGs) revealing that 77 Wzy polymerases were classified into 56 HGs, which indicate the high specificity of wzy between different types. Accordingly, wzy-based capsular genotyping could differentiate capsule types except for those lacking wzy (K29 and K50), those sharing identical wzy (K22 vs. K37); and should be carefully applied in those exhibited high similarity (K12 vs. K41, K2 vs. K13, K74 vs. K80, K79 vs. KN1 and K30 vs. K69). Comparison of CPS structures in several capsular types that shared similarity in their gene contents implies possible functions of glycosyltransferases. Therefore, our results provide complete set of cps in various types of Klebsiella spp., which enable the understandings of relationship between genes and CPS structures and are useful for identification of documented or new capsular types

Capsular Polysaccharide Synthesis Regions in Klebsiella Pneumoniae Serotype K57 and a New Capsular Serotype

Community-acquired pyogenic liver abscess caused by Klebsiella pneumoniae is an emerging infectious disease. We explored the capsular polysaccharide synthesis (cps) regions of three non-K1, non-K2 K. pneumoniae strains, A1142, A7754 , and A1517, from Taiwanese patients experiencing pyogenic liver abscess. Two of the strains, A1142 and A7754, belonged to capsular serotype K57, while the third belonged to a new capsular serotype, different from the previously reported 77 serotypes. Deletion and complementation experiments suggested that a unique K57 gene, a homologue of wzy, was essential for K57 capsular synthesis and confirmed that this gene cluster was a genetic coding region for K57. Compared to K1 and K2 strains, the three strains were all serum sensitive, suggesting that host factors might also be involved in the three patients . PCR using primers from specific genes for K57 was more sensitive and specific than traditional serotyping. The remaining strain, A1517, did not react to the antisera from any of the 77 serotypes, and none of the 77 reference strains reacted to the serum against this strain. Moreover, PCR analyses using various primer pairs from the serotype-specific open reading frames did not reveal cross-reactivity to any of the 77 reference strains, suggesting that this strain likely represents a new capsular type . We conclude that sequences from these two cps regions are very useful in detecting K57 and the new cps genotype

Identification of Capsular Types in Carbapenem-Resistant Klebsiella pneumoniae Strains by wzc Sequencing and Implications for Capsule Depolymerase Treatment

Klebsiella pneumoniae is an important human pathogen associated with a variety of diseases, and the prevalence of multidrug-resistant K. pneumoniae (MDRKP) is rapidly increasing. Here we determined the capsular types of 85 carbapenem-resistant K. pneumoniae (CRKP) strains by wzc sequencing and investigated the presence of carbapenemases and integrons among CRKP strains. Ten CRKP strains (12%) were positive for carbapenemase (imipenemase, 6/85 strains; K. pneumoniae carbapenemase, 3/85 strains; Verona integron-encoded metallo-beta-lactamase, 1/85 strains). Capsular type K64 accounted for 32 CRKP strains (38%), followed by K62 (13%), K24 (8%), KN2 (7%), and K28 (6%). Sequence types (STs) were determined by multilocus sequence typing (MLST), and the results indicated that ST11, which accounted for 47% of these CRKP strains (40/85 strains), was the major ST. We further isolated a K64-specific capsule depolymerase (K64dep), which could enhance serum and neutrophil killing in vitro and increase survival rates for K64 K. pneumoniae-inoculated mice. The toxicity study demonstrated that mice treated with K64dep showed normal biochemical parameters and no significant histopathological changes of liver, kidney, and spleen, indicating that enzyme treatment did not cause toxicity in mice. Therefore, the findings of capsular type clustering among CRKP strains and effective treatment with capsule depolymerase for MDRKP infections are important for capsule-based vaccine development and therapy

An evaluative study of test items of geography subject in the Basic Competence Test and basic abilities of geography subject for junior high school students in Taiwan

[[abstract]]The purpose of this study is to understand the basic geographic abilities and knowledge of Taiwanese junior high graduates by analyzing their performance in the social studies- geography of The Basic Competence Test for junior high school students. According to the distribution of examination questions, calculating the percentage of the knowledge dimension and ability dimension detailed items that more than 50% graduates can pass them. If the percentage is more than 80% were regarded as the equipped abilities and knowledge of the graduates. According to the results of researching, 1. As to the distribution of examination questions in the ability dimension, there were the most examination questions in the understanding sub-dimension, followed by the remembering. Few questions were in the checking sub-dimension and none was in planning. In knowledge dimension, there were the most questions in the “the characteristic of regions” sub-dimension, followed by “the analyzing of space”, “geographic skills” and “environmental geography”. 2. As to the passing rates, graduates had better performance in “remembering”、 “understanding”、 “applying” abilities sub-dimensions, and better performance in “geographic skills”、 “the analyzing of space”、 “the characteristic of regions” knowledge sub-dimensions. 3. graduates had the geographic basic ability of understanding in the ability dimension were “understanding” and in the knowledge dimension were “geographic skills”、 “the analyzing of space” and “environmental geography”. If the graduates were grouped into non-taking “Grade 1-9 Curriculum”（2001-2004） and taking “Grade 1-9 Curriculum”（2005）, in 2001-2004, graduates had only the “understanding” ability in ability dimension and “geographic skills”、 “the analyzing of space” in the knowledge dimension. In 2005, graduates had “understanding”、 “applying” ability in ability dimension and “geographic skills”、 “the analyzing of space”、 “the characteristic of regions”、 “environmental geography” in the knowledge dimension. Further discussion and suggestion were also mentioned.

Colistin Resistance Mechanisms in Klebsiella pneumoniae Strains from Taiwan

Colistin is one of the antibiotics of last resort for the treatment of carbapenem-resistant Klebsiella pneumoniae infection. This study showed that capsular type K64 (50%) and ST11 (53.9%) are the prevalent capsular and sequence types in the colistin-resistant strains in Taiwan. The interruption of transcripts (38.5%) and amino acid mutation (15.4%) in mgrB are the major mechanisms contributing to colistin resistance. In addition, novel single amino acid changes in MgrB (Stop48Tyr) and PhoQ (Leu26Pro) were observed to contribute to colistin resistance

Use of a Dictyostelium Model for Isolation of Genetic Loci Associated with Phagocytosis and Virulence in Klebsiella pneumoniae▿

Phagocytosis resistance is an important virulence factor in Klebsiella pneumoniae. Dictyostelium has been used to study the interaction between phagocytes and bacteria because of its similarity to mammalian macrophages. In this study, we used a Dictyostelium model to investigate genes for resistance to phagocytosis in NTUH-K2044, a strain of K. pneumoniae causing pyogenic liver abscess that is highly resistant to phagocytosis. A total of 2,500 transposon mutants were screened by plaque assay, and 29 of them permitted phagocytosis by Dictyostelium. In the 29 mutants, six loci were identified; three were capsular synthesis genes. Of the other three, one was related to carnitine metabolism, one encoded a subunit of protease (clpX), and one encoded a lipopolysaccharide O-antigen transporter (wzm). Deletion and complementation of these genes showed that only ΔclpX and Δwzm mutants became susceptible to Dictyostelium phagocytosis, and their complementation restored the phagocytosis resistance phenotype. These two mutants were also susceptible to phagocytosis by human neutrophils and revealed attenuated virulence in a mouse model, implying that they play important roles in the pathogenesis of K. pneumoniae. Furthermore, we demonstrated that clpP, which exists in an operon with clpX, was also involved in resistance to phagocytosis. The transcriptional profile of ΔclpX was examined by microarray analysis and revealed a 3-fold lower level of expression of capsular synthesis genes. Therefore, we have identified genes involved in resistance to phagocytosis in K. pneumoniae using Dictyostelium, and this model is useful to explore genes associated with resistance to phagocytosis in heavily encapsulated bacteria

Molecular Characterization of Putative Virulence Determinants in Burkholderia pseudomallei

The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Using a virulent clinical isolate of B. pseudomallei and an attenuated strain of the same B. pseudomallei isolate, 6 genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BURPS1710A_1419 were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional gene BPSL3147 that had been identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and were confirmed by PCR. BPSL2033::Km and BURPS1710A_1419::Km mutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model. BPSL2033::Km demonstrated weak statistical significance (P=0.049) at 8 hours after infection in macrophage infection study but this was not seen in BURPS1710A_1419::Km. Nevertheless, complemented strains of both genes were able to partially restore the gene defects in both in vitro and in vivo studies, thus suggesting that they individually play a minor role in the virulence of B. pseudomallei