Transmission and evolution of OXA-48-producing Klebsiella pneumoniae ST11 in a single hospital in Taiwan.

OBJECTIVES: Epidemic spread of OXA-48-producing Klebsiella pneumoniae, mainly mediated by the transmission of a blaOXA-48-carrying plasmid, has threatened global health during the last decade. Since its introduction to Taiwan in 2013, OXA-48 has become the second most common carbapenemase. We described the transmission and evolution of an OXA-producing K. pneumoniae clone in a single hospital. METHODS: Twenty-two OXA-48 K. pneumoniae were isolated between October 2013 and December 2015. Comparative genomic analysis was performed based on the WGS data generated with Illumina and MinION techniques. RESULTS: Seventeen of the 22 OXA-48 K. pneumoniae that belonged to ST11, with the same capsular genotype, KL64, and differed from each other by seven or fewer SNPs, were considered outbreak strains. Eight of the 17 outbreak strains harboured a 65499 bp blaOXA-48-carrying IncL plasmid (called pOXA48). pOXA48 was absent from the remaining nine strains. Instead, a 24.9 kb blaOXA-48-carrying plasmid fragment was integrated into a prophage region of their chromosomes. Transmission routes of the ST11_KL64 K. pneumoniae sublineages, which carried either pOXA48 or chromosomally integrated blaOXA-48, were reconstructed. CONCLUSIONS: Clonal expansion of ST11_KL64 sublineages contributed to the nosocomial outbreak of OXA-48 K. pneumoniae. The chromosome-borne blaOXA-48 lineage emerged during a 2 year period in a single hospital. Dissemination of OXA-48, which is vertically transmitted in K. pneumoniae even in the absence of selective pressure from antimicrobials, deserves public health attention.

2,4-Diamino-Quinazoline, a Wnt Signaling Inhibitor, Suppresses Gastric Cancer Progression and Metastasis.

Gastric cancer (GC) is among the most treatment-refractory epithelial malignancies. Aberrant activation of Wnt/ß-catenin-signaling has been implicated in a variety of human cancers, including gastric cancer. Here we report that the elevated expression of lymphoid enhancer binding factor 1 (Lef1) is associated with the TNM (tumor- node-metastasis) stage of gastric cancer. Subsequently, 2,4-diamino-quinazoline (2,4-DAQ), a selective inhibitor of Lef1, was identified to suppress the expression of Wnt/ß-catenin target genes such as AXIN2, MYC and LGR5 and result in the suppression of gastric cancer cell growth through the apoptotic pathway. The 2,4-DAQ also exhibited an inhibitory effect on the migration/invasion of gastric cancer cells. Importantly, the treatment of human gastric tumor xenograft with 2,4-DAQ suppressed tumor growth in a nude mouse model. Furthermore, 2,4-DAQ appears effective on patient-derived organoids (PDOs). Transcriptome sequencing analysis also revealed that 2,4-DAQ are more effective on the gastric cancers that exhibit higher expression levels of Wnt-signaling pathway-related genes than their adjacent normal gastric tissues.

Inhibition of CCAR1, a Coactivator of ß-Catenin, Suppresses the Proliferation and Migration of Gastric Cancer Cells.

The aberrant activation of Wnt signaling has been implicated in a variety of human cancers, including gastric cancer. Given the current hypothesis that cancer arises from cancer stem cells (CSCs), targeting the critical signaling pathways that support CSC self-renewal appears to be a useful approach for cancer therapy. Cell cycle and apoptosis regulator 1 (CCAR1) is a transcriptional coactivator which has been shown to be a component of Wnt/ß-catenin signaling, and which plays an important role in transcriptional regulation by ß-catenin. However, the function and clinical significance of CCAR1 in gastric cancer have not been elucidated. Here, we show that elevated CCAR1 nuclear expression correlates with the occurrence of gastric cancer. In addition, RNAi-mediated CCAR1 reduction not only suppressed the cell growth and increased apoptosis in AGS and MKN28 cells, but also reduced the migration and invasion ability of these cells. Furthermore, an in vivo xenograft assay revealed that the expression level of CCAR1 was critical for tumorigenesis. Our data demonstrates that CCAR1 contributes to carcinogenesis in gastric cancer and is required for the survival of gastric cancer cells. Moreover, CCAR1 may serve as a diagnostic marker and a potential therapeutic target.

The role of pgaC in Klebsiella pneumoniae virulence and biofilm formation.

BACKGROUND: Klebsiella pneumoniae has emerged as one of the major pathogens for community-acquired and nosocomial infections. A four-gene locus that had a high degree similarity with Escherichia coli pgaABCD and Yersinia pestis hmsHFRS was identified in K. pneumoniae genomes. The pgaABCD in E. coli encodes the envelope-spanning Pga machinery for the synthesis and secretion of poly-ß-linked N-acetylglucosamine (PNAG). In a limited number of phylogenetically diverse bacteria, PNAG was demonstrated to mediate biofilm formation and had a role in the host-bacteria interactions. The presence of conserved pgaABCD locus among various K. pneumoniae strains suggested a putative requirement of PNAG for this bacterium. RESULTS: In this study, an in-frame deletion of pgaC was generated in K. pneumoniae CG43 and named ΔpgaC. The loss of pgaC affected the production of PNAG and attenuated the enhancement of in vitro biofilm formation upon the addition of bile salts mixture. In mouse models, ΔpgaC exhibited a weakened ability to colonize the intestine, to disseminate extraintestinally, and to induce a systemic infection when compared to K. pneumoniae CG43. CONCLUSIONS: Our study demonstrated that pgaC participated in the bile salts induced biofilm formation and was required for K. pneumoniae virulence in vivo.

Arecoline downregulates levels of p21 and p27 through the reactive oxygen species/mTOR complex 1 pathway and may contribute to oral squamous cell carcinoma.

Arecoline, the major alkaloid of areca nut, has been shown to cause strong genotoxicity and is considered a potential carcinogen. However, the detailed mechanism for arecoline-induced carcinogenesis remains obscure. In this study, we noticed that the levels of p21 and p27 increased in two oral squamous cell carcinoma cell lines with high confluence. Furthermore, when treated with arecoline, elevated levels of p21 and p27 could be downregulated through the reactive oxygen species/mTOR complex 1 (ROS/mTORC1) pathway. Although arecoline decreased the activity of mTORC1, the amounts of autophagosome-like vacuoles or type II LC3 remained unchanged, suggesting that the downregulation of p21 and p27 was independent of autophagy-mediated protein destruction. Arecoline also caused DNA damage through ROS, indicating that the reduced levels of p21 and p27 might facilitate G (1) /S transition of the cell cycle and subsequently lead to error-prone DNA replication. In conclusion, these data have provided a possible mechanism for arecoline-induced carcinogenesis in subcytolytic doses in vivo.

CCAR1 is required for Ngn3-mediated endocrine differentiation.

Neurogenin3 (Ngn3) is a basic helix-loop-helix transcription factor that specifies pancreatic endocrine cell fates during pancreas development. It can also initiate a transdifferentiation program when expressed in pancreatic exocrine and ductal cells. However, how Ngn3 initiates a transcriptional cascade to achieve endocrine differentiation is still poorly understood. Here, we show that cell cycle and apoptosis regulator 1 (CCAR1), which is a transcriptional coactivator for nuclear receptors, also interacts with Ngn3. The association between Ngn3 and CCAR1 was verified by pull-down assays and co-immunoprecipitation analyses. Using gene reporter assays, we found that CCAR1 is essential for Ngn3 to activate the expression of the reporter genes containing the NeuroD promoter. Moreover, down-regulation of endogenous CCAR1 in the PANC-1 pancreatic ductal cell line inhibits the transdifferentiation program initiated by Ngn3. CCAR1 is, therefore, a novel partner of Ngn3 in mediating endocrine differentiation.

Reversine suppresses oral squamous cell carcinoma via cell cycle arrest and concomitantly apoptosis and autophagy.

BACKGROUND: The effective therapies for oral cancer patients of stage III and IV are generally surgical excision and radiation combined with adjuvant chemotherapy using 5-Fu and Cisplatin. However, the five-year survival rate is still less than 30% in Taiwan. Therefore, evaluation of effective drugs for oral cancer treatment is an important issue. Many studies indicated that aurora kinases (A, B and C) were potential targets for cancer therapies. Reversine was proved to be a novel aurora kinases inhibitor with lower toxicity recently. In this study, the potentiality for reversine as an anticancer agent in oral squamous cell carcinoma (OSCC) was evaluated. METHODS: Effects of reversine on cell growth, cell cycle progress, apoptosis, and autophagy were evaluated mainly by cell counting, flow cytometry, immunoblot, and immunofluorescence. RESULTS: The results demonstrated that reversine significantly suppressed the proliferation of two OSCC cell lines (OC2 and OCSL) and markedly rendered cell cycle arrest at G2/M stage. Reversine also induced cell death via both caspase-dependent and -independent apoptosis. In addition, reversine could inhibit Akt/mTORC1 signaling pathway, accounting for its ability to induce autophagy. CONCLUSIONS: Taken together, reversine suppresses growth of OSCC via multiple mechanisms, which may be a unique advantage for developing novel therapeutic regimens for treatment of oral cancer in the future.

Assessment of hypermucoviscosity as a virulence factor for experimental Klebsiella pneumoniae infections: comparative virulence analysis with hypermucoviscosity-negative strain.

BACKGROUND: Klebsiella pneumoniae displaying the hypermucoviscosity (HV) phenotype are considered more virulent than HV-negative strains. Nevertheless, the emergence of tissue-abscesses-associated HV-negative isolates motivated us to re-evaluate the role of HV-phenotype. RESULTS: Instead of genetically manipulating the HV-phenotype of K. pneumoniae, we selected two clinically isolated K1 strains, 1112 (HV-positive) and 1084 (HV-negative), to avoid possible interference from defects in the capsule. These well-encapsulated strains with similar genetic backgrounds were used for comparative analysis of bacterial virulence in a pneumoniae or a liver abscess model generated in either naïve or diabetic mice. In the pneumonia model, the HV-positive strain 1112 proliferated to higher loads in the lungs and blood of naïve mice, but was less prone to disseminate into the blood of diabetic mice compared to the HV-negative strain 1084. In the liver abscess model, 1084 was as potent as 1112 in inducing liver abscesses in both the naïve and diabetic mice. The 1084-infected diabetic mice were more inclined to develop bacteremia and had a higher mortality rate than those infected by 1112. A mini-Tn5 mutant of 1112, isolated due to its loss of HV-phenotype, was avirulent to mice. CONCLUSION: These results indicate that the HV-phenotype is required for the virulence of the clinically isolated HV-positive strain 1112. The superior ability of the HV-negative stain 1084 over 1112 to cause bacteremia in diabetic mice suggests that factors other than the HV phenotype were required for the systemic dissemination of K. pneumoniae in an immunocompromised setting.

The high incidence of JC virus infection in urothelial carcinoma tissue in Taiwan.

Human polyomaviruses, JC virus (JCV) and BK virus (BKV), usually remain latent in kidney and urothelial tissue after primary infection. Infection with human polyomavirus has still not been correlated conclusively with malignancy of kidney and urothelial tissue. The present study investigated further the possible relationship between JCV/BKV infection and urothelial carcinoma. Tissue samples were examined from 33 urothelial carcinomas and 5 renal cell carcinomas for JCV/BKV infection, using nested PCR with primers common to both JCV and BKV. The viral genotypes were further verified by endonuclease digestion and DNA sequencing following the PCR. In addition, immunohistochemistry and Western blotting were also performed to detect viral large tumor protein (LT) and the late capsid protein (VP1) in the tissue samples. The results from nested PCR showed that 90.1% (30/33) of the urothelial carcinomas samples and all of the renal cell carcinomas samples (5/5) were JCV DNA positive. Both archetypal and re-arranged JCV genotypes were detected. On the other hand, BKV DNA was detected in only one (3%) of the urothelial carcinoma tissue samples. The immunohistochemical results showed that 30% (10/33) of urothelial carcinoma tissues was stained positive for large tumor antigen (LT). However, the structural protein VP1 was not detectable in any of the tissue samples examined. The present study demonstrated that JCV is highly prevalent in urothelial carcinoma tissue as is the expression of large tumor antigen. Therefore, the findings support the hypothesis that JCV infection is associated with urothelial carcinoma.

Two ST11 Klebsiella pneumoniae strains exacerbate colorectal tumorigenesis in a colitis-associated mouse model.

Sequence type (ST) 11 is one of the major lineages of carbapenem-resistant Klebsiella pneumoniae (CRKP). Although the gastrointestinal (GI) carriage of CRKP predisposes individuals to subsequent infections, little is known for its impact on gut homeostasis. In this study, we investigated the association between ST11 CRKP colonization and colorectal cancer (CRC). Two ST11 CRKP, KPC160111 (KL47) and KPC160132 (KL64), were selected as the representative strains. We used azoxymethane (AOM) and dextran sodium sulfate (DSS) to initiate a colitis-associated CRC model. Both strains established prolonged colonization in the GI tract of the AOM-DSS-treated BALB/c mice and aggravated gut dysbiosis. Under this AOM-DSS-induced setting, ST11 K. pneumoniae colonization significantly promoted the growth and progression of colorectal adenomas to high-grade dysplasia. Numerous crypts were formed inside the enlarged adenomas, in which CD163+ tumor-associated macrophages accumulated. Similarly, ST11 K. pneumoniae also increased the population size of the CD163+ macrophages with the M2 phenotype in the peritoneal cavity of LPS-primed BALB/c mice. When applied to RAW264.7 cells, ST11 K. pneumoniae polarized the macrophages toward an M2 phenotype through the inhibition of IKK-NFκB and the activation of STAT6-KLF4-IL-10. Through the M2-skewing ability, ST11 K. pneumoniae promoted the accumulation of CD163+ macrophages in the adenomatous crypts to create an immunosuppressive niche, which not only accommodated the extended stay for its own sake but also deteriorated colorectal tumorigenesis.

Epigenetic Silencing of STAT3-Targeted miR-193a, by Constitutive Activation of JAK/STAT Signaling, Leads to Tumor Progression Through Overexpression of YWHAZ in Gastric Cancer.

PURPOSE: The purpose of this study was to identify genes that were epigenetically silenced by STAT3 in gastric cancer. METHODS: MBDcap-Seq and expression microarray were performed to identify genes that were epigenetically silenced in AGS gastric cancer cell lines depleted of STAT3. Cell lines and animal experiments were performed to investigate proliferation and metastasis of miR-193a and YWHAZ in gastric cancer cell lines. Bisulfite pyrosequencing and tissue microarray were performed to investigate the promoter methylation of miR-193a and expression of STAT3, YWHAZ in patients with gastritis (n = 8) and gastric cancer (n = 71). Quantitative methylation-specific PCR was performed to examine miR-193a promoter methylation in cell-free DNA of serum samples in gastric cancer patients (n = 19). RESULTS: As compared with parental cells, depletion of STAT3 resulted in demethylation of a putative STAT3 target, miR-193a, in AGS gastric cancer cells. Although bisulfite pyrosequencing and epigenetic treatment confirmed that miR-193a was epigenetically silenced in gastric cancer cell lines, ChIP-PCR found that it may be indirectly affected by STAT3. Ectopic expression of miR-193a in AGS cells inhibited proliferation and migration of gastric cancer cells. Further expression microarray and bioinformatics analysis identified YWHAZ as one of the target of miR-193a in AGS gastric cancer cells, such that depletion of YWHAZ reduced migration in AGS cells, while its overexpression increased invasion in MKN45 cells in vitro and in vivo. Clinically, bisulfite pyrosequencing revealed that promoter methylation of miR-193a was significantly higher in human gastric cancer tissues (n = 11) as compared to gastritis (n = 8, p < 0.05). Patients infected with H. pylori showed a significantly higher miR-193a methylation than those without H. pylori infection (p < 0.05). Tissue microarray also showed a positive trend between STAT3 and YWHAZ expression in gastric cancer patients (n = 60). Patients with serum miR-193a methylation was associated with shorter overall survival than those without methylation (p < 0.05). CONCLUSIONS: Constitutive activation of JAK/STAT signaling may confer epigenetic silencing of the STAT3 indirect target and tumor suppressor microRNA, miR-193a in gastric cancer. Transcriptional suppression of miR-193a may led to overexpression of YWHAZ resulting in tumor progression. Targeted inhibition of STAT3 may be a novel therapeutic strategy against gastric cancer.

Genetic requirements for Klebsiella pneumoniae-induced liver abscess in an oral infection model.

Klebsiella pneumoniae is the predominant pathogen of primary liver abscess. However, our knowledge regarding the molecular basis of how K. pneumoniae causes primary infection in the liver is limited. We established an oral infection model that recapitulated the characteristics of liver abscess and conducted a genetic screen to identify the K. pneumoniae genes required for the development of liver abscess in mice. Twenty-eight mutants with attenuated growth in liver or spleen samples out of 2,880 signature-tagged mutants that produced the wild-type capsule were identified, and genetic loci which were disrupted in these mutants were identified to encode products with roles in cellular metabolism, adhesion, transportation, gene regulation, and unknown functions. We further evaluated the virulence attenuation of these mutants in independent infection experiments and categorized them accordingly into three classes. In particular, the class I and II mutant strains exhibited significantly reduced virulence in mice, and most of these strains were not detected in extraintestinal tissues at 48 h after oral inoculation. Interestingly, the mutated loci of about one-third of the class I and II mutant strains encode proteins with regulatory functions, and the transcript abundances of many other genes identified in the same screen were markedly changed in these regulatory mutant strains, suggesting a requirement for genetic regulatory networks for translocation of K. pneumoniae across the intestinal barrier. Furthermore, our finding that preimmunization with certain class I mutant strains protected mice against challenge with the wild-type strain implied a potential application for these strains in prophylaxis against K. pneumoniae infections.

Single-cell transcript analysis of pancreas development.

DNA microarray analysis was combined with a modified single-cell PCR procedure to study gene expression profiles of single cells at different stages of pancreatic development. This method identifies distinct cell types at embryonic day 10.5, a stage when the pancreatic epithelium is morphologically uniform. Some cells express unexpected combinations of genes, and these expression patterns provide new insights into pancreas development. Following on these findings, we use PCR products from different cell types to identify novel pancreatic genes, some of which mark subtypes of developing pancreatic cells. By integrating these data with previous genetic and biochemical studies, we propose a pathway for pancreatic cell development. This form of single-cell transcriptional analysis can be applied to any developmental process or tissue to characterize distinct cell types.

Clonal dissemination of carbapenemase-producing Klebsiella pneumoniae: Two distinct sub-lineages of Sequence Type 11 carrying blaKPC-2 and blaOXA-48.

OBJECTIVES: The global spread of carbapenem-resistant Klebsiella pneumoniae (CR-Kp) has become a massive threat to human health. We investigated the clonal relatedness of CR-Kp strains in central Taiwan. METHODS: CR-Kp strains were prospectively collected from inpatients referred to Chung Shan Medical University Hospital (CSMUH) during September 2011 to December 2015. The presence of carbapenemase genes, including blaKPC-2, blaVIM-1, blaNDM-1, and blaOXA-48, was analysed with polymerase chain reaction (PCR) and sequence determination. Clonal relatedness was determined by pulse-field gel electrophoresis and multilocus sequence typing. Capsule synthesis loci were typed based on the variation of the wzi gene. RESULTS: A total of 174 CR-Kp strains were collected. KPC-2 and OXA-48 were present in 63 (36.2%) and 22 (12.6%) CR-Kp strains, respectively. Two strains isolated in 2014 coproduced KPC-2 and OXA-48. Nearly all (98%) carbapenemase-producing K. pneumoniae strains belonged to the ST11 clone and could be further grouped into distinct sub-lineages. Intriguingly, the first sub-lineage, designated ST11-Clade I, contained all KPC-2 strains; OXA-48 strains were mostly included in the second sub-lineage, ST11-Clade II. Furthermore, a variation on the capsule synthesis loci was detected between these two sub-lineages: KL-47 was assigned to ST11-Clade I, whereas KL-64 or KL-9 were the main types for the ST11-Clade II strains. CONCLUSIONS: Clonal expansion of ST11 was responsible for the dissemination of carbapenemase-producing K. pneumoniae. Although KPC-2 still predominates, OXA-48 has emerged rapidly. Co-existence of KPC-2 and OXA-48 in two ST11-Clade I K. pneumoniae highlights the urgency to unravel mechanisms that contribute to this highly transmissible lineage.

Rhodiolae Kirliowii Radix et Rhizoma and Crataegus pinnatifida Fructus Extracts Effectively Inhibit BK Virus and JC Virus Infection of Host Cells.

The human polyomaviruses BK (BKPyV) and JC (JCPyV) are ubiquitous pathogens long associated with severe disease in immunocompromised individuals. BKPyV causes polyomavirus-associated nephropathy and hemorrhagic cystitis, whereas JCPyV is the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy. No effective therapies targeting these viruses are currently available. The goal of this study was to identify Chinese medicinal herbs with antiviral activity against BKPyV and JCPyV. We screened extracts of Chinese medicinal herbs for the ability to inhibit hemagglutination by BKPyV and JCPyV virus-like particles (VLPs) and the ability to inhibit BKPyV and JCPyV binding and infection of host cells. Two of the 40 herbal extracts screened, Rhodiolae Kirliowii Radix et Rhizoma and Crataegus pinnatifida Fructus, had hemagglutination inhibition activity on BKPyV and JCPyV VLPs and further inhibited infection of the cells by BKPyV and JCPyV, as evidenced by reduced expression of viral proteins in BKPyV-infected and JCPyV-infected cells after treatment with Rhodiolae Kirliowii Radix et Rhizoma or Crataegus pinnatifida Fructus extract. The results in this work show that both Rhodiolae Kirliowii Radix et Rhizoma and Crataegus pinnatifida Fructus may be sources of potential antiviral compounds for treating BKPyV and JCPyV infections.

Colibactin Contributes to the Hypervirulence of pks+ K1 CC23 Klebsiella pneumoniae in Mouse Meningitis Infections.

Klebsiella pneumoniae is the most common pathogen of community-acquired meningitis in Taiwan. However, the lack of a physiologically relevant meningitis model for K. pneumoniae has impeded research into its pathogenesis mechanism. Based on the core genome MLST analyses, the hypervirulent K1 K. pneumoniae strains, which are etiologically implicated in adult meningitis, mostly belong to a single clonal complex, CC23. Some K1 CC23 K. pneumoniae strains carry a gene cluster responsible for colibactin production. Colibactin is a small genotoxic molecule biosynthesized by an NRPS-PKS complex, which is encoded by genes located on the pks island. Compared to other hypervirulent K. pneumoniae which primarily infect the liver, the colibactin-producing (pks+) K1 CC23 strains had significant tropism toward the brain of BALB/c mice. We aimed in this study to develop a physiologically relevant meningitis model with the use of pks+ K1 CC23 K. pneumoniae. Acute meningitis was successfully induced in adult BALB/c male mice through orogastric, intranasal, and intravenous inoculation of pks+ K1 CC23 K. pneumoniae. Besides the typical symptoms of bacterial meningitis, severe DNA damages, and caspase 3-independent cell death were elicited by the colibactin-producing K1 CC23 K. pneumoniae strain. The deletion of clbA, which abolished the production of colibactin, substantially hindered K. pneumoniae hypervirulence in the key pathogenic steps toward the development of meningitis. Our findings collectively demonstrated that colibactin was necessary but not sufficient for the meningeal tropism of pks+ K1 CC23 K. pneumoniae, and the mouse model established in this study can be applied to identify other virulence factors participating in the development of this life-threatening disease.

TCTP is essential for ß-cell proliferation and mass expansion during development and ß-cell adaptation in response to insulin resistance.

The perinatal period is critical for ß-cell mass establishment, which is characterized by a transient burst in proliferation to increase ß-cell mass in response to the need for glucose homeostasis throughout life. In adulthood, the ability of ß-cells to grow, proliferate, and expand their mass is also characteristic of pathological states of insulin resistance. Translationally controlled tumor-associated protein (TCTP), an evolutionarily highly conserved protein that is implicated in cell growth and proliferation, has been identified as a novel glucose-regulated survival-supporting protein in pancreatic ß-cells. In this study, the enhanced ß-cell proliferation detected both during the perinatal developmental period and in insulin-resistant states in high-fat diet-fed mice was found to parallel the expression of TCTP in pancreatic ß-cells. Specific knockout of TCTP in ß-cells led to increased expression of total and nuclear Forkhead box protein O1 and tumor suppressor protein 53, and decreased expression of p70S6 kinase phosphorylation and cyclin D2 and cyclin-dependent kinase 2. This resulted in decreased ß-cell proliferation and growth, reduced ß-cell mass, and insulin secretion. Together, these effects led to hyperglycemia. These observations suggest that TCTP is essential for ß-cell mass expansion during development and ß-cell adaptation in response to insulin resistance.

Genotoxic Klebsiella pneumoniae in Taiwan.

BACKGROUND: Colibactin is a nonribosomal peptide-polyketide synthesized by multi-enzyme complexes encoded by the pks gene cluster. Colibactin-producing Escherichia coli have been demonstrated to induce host DNA damage and promote colorectal cancer (CRC) development. In Taiwan, the occurrence of pyogenic liver abscess (PLA) has been suggested to correlate with an increasing risk of CRC, and Klebsiella pneumoniae is the predominant PLA pathogen in Taiwan. METHODOLOGY/PRINCIPAL FINDINGS: At the asn tRNA loci of the newly sequenced K. pneumoniae 1084 genome, we identified a 208-kb genomic island, KPHPI208, of which a module identical to the E. coli pks colibactin gene cluster was recognized. KPHPI208 consists of eight modules, including the colibactin module and the modules predicted to be involved in integration, conjugation, yersiniabactin production, microcin production, and unknown functions. Transient infection of BALB/c normal liver cells with K. pneumoniae 1084 increased the phosphorylation of histone H2AX, indicating the induction of host DNA damage. Colibactin was required for the genotoxicity of K. pneumoniae 1084, as it was diminished by deletion of clbA gene and restored to the wild type level by trans-complementation with a clbA coding plasmid. Besides, BALB/c mice infected with K. pneumoniae 1084 exhibited enhanced DNA damage in the liver parenchymal cells when compared to the isogenic clbA deletion mutant. By PCR detection, the prevalence of pks-positive K. pneumoniae in Taiwan is 25.6%, which is higher than that reported in Europe (3.5%), and is significantly correlated with K1 type, which predominantly accounted for PLA in Taiwan. CONCLUSIONS: Our knowledge regarding how bacteria contribute to carcinogenesis has just begun. The identification of genotoxic K. pneumoniae and its genetic components will facilitate future studies to elucidate the molecular basis underlying the link between K. pneumoniae, PLA, and CRC.