Pei-Fang Hsieh

Serum-Induced Iron-Acquisition Systems and TonB Contribute to Virulence in Klebsiella pneumoniae Causing Primary Pyogenic Liver Abscess

BACKGROUND Klebsiella pneumoniae has become the predominant pathogen causing primary pyogenic liver abscess (PLA). METHODS K. pneumoniae was stimulated by human serum, and gene expression was analyzed by microarray. RESULTS Three putative iron acquisition systems, Yersinia high-pathogenicity island (HPI), iucABCDiutA, and iroA(iroNDCB), that increased in expression and predominated in PLA-associated K. pneumoniae strains were identified. By use of siderophore uptake assays, these 3 systems were confirmed to be siderophore-dependent iron acquisition systems. Only the irp2-iuc-iroA triple mutant showed decreased virulence in mice. Full-genome analysis of K. pneumoniae strain NTUH-K2044 identified 10 putative iron uptake systems. Seven of these 10 systems were TonB dependent, including Yersinia HPI, iucABCDiutA, and iroA. A tonB deletion mutant was demonstrated to have profound attenuation of virulence. Immunization with the tonB mutant resulted in seroconversion of extracellular polysaccharide antibodies and protective efficacy against subsequent exposure to the parental strain. CONCLUSIONS Iron uptake systems were the genes in K. pneumoniae that were highly up-regulated in response to sera. Although there are multiple iron transporter systems in NTUH-K2044, a mutation in all 3 loci (irp2, iuc, and iroA) is necessary to decrease virulence. The tonB mutant is a potential vaccine candidate because it can induce a significant protective immune response against challenge with a wild-type strain.

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

ABSTRACT 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.

Isolation of Genes Involved in Biofilm Formation of a Klebsiella pneumoniae Strain Causing Pyogenic Liver Abscess

Background Community-acquired pyogenic liver abscess (PLA) complicated with meningitis and endophthalmitis caused by Klebsiella pneumoniae is an emerging infectious disease. To investigate the mechanisms and effects of biofilm formation of K. pneumoniae causing PLA, microtiter plate assays were used to determine the levels of biofilm formed by K. pneumoniae clinical isolates and to screen for biofilm-altered mutants from a transposon mutant library of a K. pneumoniae PLA-associated strain. Methodology/Principal Findings The biofilm formation of K. pneumoniae was examined by microtiter plate assay. Higher levels of biofilm formation were demonstrated by K. pneumoniae strains associated with PLA. A total of 23 biofilm-decreased mutants and 4 biofilm-increased mutants were identified. Among these mutants, a biofilm-decreased treC mutant displayed less mucoviscosity and produced less capsular polysaccharide (CPS), whereas a biofilm-increased sugE mutant displayed higher mucoviscosity and produced more CPS. The biofilm phenotypes of treC and sugE mutants also were confirmed by glass slide culture. Deletion of treC, which encodes trehalose-6-phosphate hydrolase, impaired bacterial trehalose utilization. Addition of glucose to the culture medium restored the capsule production and biofilm formation in the treC mutant. Transcriptional profile analysis suggested that the increase of CPS production in ΔsugE may reflect elevated cps gene expression (upregulated through rmpA) in combination with increased treC expression. In vivo competition assays demonstrated that the treC mutant strain was attenuated in competitiveness during intragastric infection in mice. Conclusions/Significance Genes important for biofilm formation by K. pneumoniae PLA strain were identified using an in vitro assay. Among the identified genes, treC and sugE affect biofilm formation by modulating CPS production. The importance of treC in gastrointestinal tract colonization suggests that biofilm formation contributes to the establishment and persistence of K. pneumoniae infection.

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 ≧94% DNA sequence identity across the variable region (CD1-VR2-CD2) of wzc. Strains with distinct K types exhibited <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.

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 microorganisms 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.

Isolation of a Bacteriophage Specific for a New Capsular Type of Klebsiella pneumoniae and Characterization of Its Polysaccharide Depolymerase

Background Klebsiella pneumoniae is one of the major pathogens causing hospital-acquired multidrug-resistant infections. The capsular polysaccharide (CPS) is an important virulence factor of K. pneumoniae. With 78 capsular types discovered thus far, an association between capsular type and the pathogenicity of K. pneumoniae has been observed. Methodology/Principal Findings To investigate an initially non-typeable K. pneumoniae UTI isolate NTUH-K1790N, the cps gene region was sequenced. By NTUH-K1790N cps-PCR genotyping, serotyping and determination using a newly isolated capsular type-specific bacteriophage, we found that NTUH-K1790N and three other isolates Ca0507, Ca0421 and C1975 possessed a new capsular type, which we named KN2. Analysis of a KN2 CPS− mutant confirmed the role of capsule as the target recognized by the antiserum and the phage. A newly described lytic phage specific for KN2 K. pneumoniae, named 0507-KN2-1, was isolated and characterized using transmission electron microscopy. Whole-genome sequencing of 0507-KN2-1 revealed a 159 991 bp double-stranded DNA genome with a G+C content of 46.7% and at least 154 open reading frames. Based on its morphological and genomic characteristics, 0507-KN2-1 was classified as a member of the Myoviridae phage family. Further analysis of this phage revealed a 3738-bp gene encoding a putative polysaccharide depolymerase. A recombinant form of this protein was produced and assayed to confirm its enzymatic activity and specificity to KN2 capsular polysaccharides. KN2 K. pneumoniae strains exhibited greater sensitivity to this depolymerase than these did to the cognate phage, as determined by spot analysis. Conclusions/Significance Here we report that a group of clinical strains possess a novel Klebsiella capsular type. We identified a KN2-specific phage and its polysaccharide depolymerase, which could be used for efficient capsular typing. The lytic phage and depolymerase also have potential as alternative therapeutic agents to antibiotics for treating K. pneumoniae infections, especially against antibiotic-resistant strains.

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.

Structure and Immunological Characterization of the Capsular Polysaccharide of a Pyrogenic Liver Abscess Caused by Klebsiella pneumoniae ACTIVATION OF MACROPHAGES THROUGH TOLL-LIKE RECEPTOR 4

The active components of a primary pyrogenic liver abscess (PLA) Klebsiella pneumoniae in stimulating cytokine expression in macrophages are still unclear. The capsular polysaccharide (CPS) of PLA K. pneumoniae is important in determining clinical manifestations, and we have shown that it consists of repeating units of the trisaccharide (→3)-β-d-Glc-(1→4)-[2,3-(S)-pyruvate]-β-d-GlcA-(1→4)-α-l-Fuc-(1→) and has the unusual feature of extensive pyruvation of glucuronic acid and acetylation of C2-OH or C3-OH of fucose. We demonstrated that PLA K. pneumoniae CPS induces secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by macrophages through Toll-like receptor 4 (TLR4) and that this effect was lost when pyruvation and O-acetylation were chemically destroyed. Furthermore, expression of TNF-α and IL-6 in PLA K. pneumoniae CPS-stimulated macrophages was shown to be regulated by the TLR4/ROS/PKC-δ/NF-κB, TLR4/PI3-kinase/AKT/NF-κB, and TLR4/MAPK signaling pathways.

Isolation of a bacteriophage and its depolymerase specific for K1 capsule of Klebsiella pneumoniae: implication in typing and treatment

BACKGROUND Klebsiella pneumoniae causing community-acquired pyogenic liver abscess complicated with metastatic meningitis and endophthalmitis has emerged recently, most frequently associated with the K1 capsular type. METHODS A bacteriophage (NTUH-K2044-K1-1) that infects K. pneumoniae NTUH-K2044 (capsular type K1) was isolated and characterized. RESULTS The phage infected all K1 strains, and none of the strains with other capsular types. Capsule deletion mutants were not lysed by this phage, suggesting that the capsule was essential for phage infection. Complete genome sequencing revealed the phage was a novel phiKMV-like virus. The gene-encoding capsule depolymerase was identified. The recombinant enzyme demonstrated specific lysis of the K1 capsule. Treatment with the phage or the recombinant enzyme provided significantly increased survival in mice infected with NTUH-K2044 strain, including one treated after the detection of a neck abscess by imaging. No obvious disease was observed after administration of this phage in mice. Phage was retained at detectable levels in liver, spleen, brain, and blood 24 hours after administration in mice. CONCLUSIONS These results demonstrate this phage and its capsule depolymerase exhibit specificity for capsular type K1 and can be used for the diagnosis and treatment of K1 K. pneumoniae infections.

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

ABSTRACT 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-β-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.