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The comorbidity of obesity and major depressive disorder (MDD) may be attributable to a bidirectional relationship and shared genetic influence. We aimed to examine the polygenic associations between obesity and MDD and to characterize their corresponding impacts on the obesity mechanism.Genome-wide genotyping was available in 106,604 unrelated individuals from Taiwan Biobank. Polygenic risk score (PRS) for body mass index (BMI) and MDD was derived to evaluate their effects on obesity-related traits. Stratified analyses were performed for the modified effect of depression on the polygenic associations.The MDD PRS was positively associated with waistline (beta in per SD increase in PRS = 0.12), hipline (beta = 0.08), waist-hip ratio (WHR) (beta = 0.05), body fat rate (beta = 0.08), BMI (beta = 0.05), overweight (OR = 1.02 for BMI ≥ 25), and obesity (OR = 1.05 for BMI ≥ 30). For the synergism between depression and BMI PRS, the presence of active depression symptoms defined by the PHQ-4 (p for interaction 0.05 for waistline, WHR, and BMI) was more salient than lifetime MDD.Limitations include recall bias for MDD due to a retrospective self-reporting questionnaire, a low response rate of the PHQ-4 for evaluating active psychological symptoms, and limited generalizability to non-Taiwanese ancestries.The shared genetic etiology of obesity and depression was demonstrated. The amplified effect of BMI polygenic effect on obesity for individuals with active depressive symptoms was also characterized. The study may be helpful for designing public health interventions to reduce the disease burden caused by obesity and depression.

Obesity has been associated with cognition in observational studies; however, whether its effect is confounding or a reverse causality remains inconclusive. This study aimed to investigate the causal relationships of overall obesity, measured by body mass index (BMI), and abdominal adiposity, measured by waist-hip ratio adjusted for BMI (WHRadjBMI), and cognition across European and Asian populations using Mendelian randomization (MR) analysis.We used publicly available genome-wide association study (GWAS) summary data of European ancestry, including BMI (n = 322,154) and WHRadjBMI (n = 210,088) from the GIANT consortium, and cognition performance (n = 257,828) from the UK Biobank and COGENT consortium. Data for individuals of Asian ancestry were retrieved from Taiwan Biobank to perform GWAS for BMI (n = 65,689), WHRadjBMI (n = 65,683), and Mini-Mental State Examination (MMSE, n = 21,273). MR analysis was carried out using the inverse-variance weighted method for the main results. Further, we examined the overall pleiotropy by MR-Egger intercept, and detected and adjusted for possible outliers using MR PRESSO.No causal effect of BMI on cognition performance (beta [95% CI] = 0.00 [-0.07, 0.07], p value = 0.91) was found for Europeans; however, a 1-SD increase in WHRadjBMI was associated with a 0.07 standardized score decrease in cognition performance (beta [95% CI] = -0.07 [-0.12, -0.02], p value = 0.006). Further, no causal effect of BMI on MMSE (beta [95% CI] = 0.01 [-0.08, 0.10], p = 0.91) was found for Asians; however, a 1-SD increase in WHRadjBMI was associated with a 0.17 standardized score decrease in MMSE (beta [95% CI] = -0.17 [-0.30, -0.03], p = 0.02). In both populations, overall pleiotropy was not detected, and outliers did not affect the robustness of the main findings.This trans-ethnic MR study reveals that abdominal adiposity, as measured by WHR adjusted for BMI, impairs cognition, whereas weak evidence suggests that BMI impairs cognition.

Psychiatric disorders, such as schizophrenia (SCZ), major depressive disorder (MDD) and bipolar disorder (BPD), are highly comorbid with type 2 diabetes. However, the mechanisms underlying such comorbidity are understudied. This study explored the familial aggregation of common psychiatric disorders and type 2 diabetes by testing family history association, and investigated the shared genetic loading between them by testing the polygenic risk score (PRS) association.A total of 105,184 participants were recruited from the Taiwan Biobank, and genome-wide genotyping data were available for 95,238 participants. The Psychiatric Genomics Consortium-derived PRS for SCZ, MDD and BPD was calculated. Logistic regression was used to estimate the OR with CIs between a family history of SCZ/MDD/BPD and a family history of type 2 diabetes, and between the PRS and the risk of type 2 diabetes.A family history of type 2 diabetes was associated with a family history of SCZ (OR 1.23, 95% CI 1.08, 1.40), MDD (OR 1.19, 95% CI 1.13, 1.26) and BPD (OR 1.26, 95% CI 1.15, 1.39). Compared with paternal type 2 diabetes, maternal type 2 diabetes was associated with a higher risk of a family history of SCZ. SCZ PRS was negatively associated with type 2 diabetes in women (OR 0.92, 95% CI 0.88, 0.97), but not in men; the effect of SCZ PRS reduced after adjusting for BMI. MDD PRS was positively associated with type 2 diabetes (OR 1.04, 95% CI 1.00, 1.07); the effect of MDD PRS reduced after adjusting for BMI or smoking. BPD PRS was not associated with type 2 diabetes.The comorbidity of type 2 diabetes with psychiatric disorders may be explained by shared familial factors. The shared polygenic loading between MDD and type 2 diabetes implies not only pleiotropy but also a shared genetic aetiology for the mechanism behind the comorbidity. The negative correlation between polygenic loading for SCZ and type 2 diabetes implies the role of environmental factors.

To elucidate the specific and shared genetic background of schizophrenia (SCZ) and bipolar disorder (BPD) to better understand their nosology, this study explored the independent and joint effects of polygenic liabilities for SCZ and BPD on cognitive aging and educational attainment among a collection of 80318 unrelated community participants from the Taiwan Biobank. Using the Psychiatric Genomics Consortium meta-analysis as a discovery sample, we calculated the polygenic risk score (PRS) for SCZ (PRSSCZ) and BPD (PRSBPD), shared PRS between SCZ and BPD (PRSSCZ+BPD), and SCZ-specific, differentiated from BPD, PRS (PRSSCZvsBPD). Based on the sign concordance of the susceptibility variants with SCZ and BPD, PRSSCZ was split into PRSSCZ_concordant and PRSSCZ_discordant and PRSBPD was split into PRSBPD_concordant and PRSBPD_discordant. Linear regression models were used to estimate the association with cognitive aging as measured by the Mini-Mental State Examination (MMSE) in individuals aged ≥ 60 years. Ordinal logistic regression models were used to estimate the association with educational attainment. PRSSCZ was negatively associated with MMSE (beta=-0.063, pBPD was positively associated with MMSE (beta=0.04, p=0.01). A larger difference between PRSSCZ and PRSBPD was associated with lower MMSE scores (beta=-0.052, pSCZ_concordant and PRSSCZ_discordant were negatively associated with MMSE scores, without a synergistic effect. There was a complex interaction between PRSBPD_concordant and PRSBPD_discordant on the MMSE scores. PRSSCZ+BPD (beta=-0.09, p=0.01) and PRSSCZvsBPD (beta=-0.13, pSCZ, PRSBPD, and PRSSCZ+BPD were positively associated with educational attainment, whereas PRSSCZvs BPD was negatively associated with educational attainment. This study provides evidence for the contrasting effect of polygenic liabilities for SCZ and BPD on cognitive aging and partially supports the hypothesis that the heterogeneity of SCZ and the positive association of polygenic liability for SCZ with education might be attributed to the shared part with BPD.

Many studies have revealed that statin therapy reduced mortality in cancer patients, especially in breast cancer, but the effect for second cancer was unclear. We, therefore, performed a comparable cohort study to determine the risk of second cancer in breast cancer patients with statin therapy. Using claims data from Taiwan’s National Health Insurance Program, this study enrolled newly diagnosed breast cancer patients from 2000 to 2007 with and without statin therapy as the statin (n = 1222) and nonstatin (n = 4888) cohorts, respectively. The nonstatin cohort was propensity score matched by cohort entry year, age, and randomly selected comorbidities. These two cohorts were followed up until the diagnosis of second cancer, death, or the end of 2011. Cox proportional hazard models were used to estimate the hazard ratios. The statin cohort had a lower incidence rate than the nonstatin cohort for second cancer (7.37 vs. 8.36 per 1000 person-years), although the difference was not significant (adjusted hazard ratio [aHR] 0.90, 95% confidence interval [CI] 0.65–1.26). Compared with the nonstatin cohort, the second cancer risk was significantly higher for patients taking pravastatin (aHR 2.71, 95% CI 1.19–6.19) but lower for those receiving multiple statin treatment (aHR 0.45, 95% CI 0.25–0.81) and combined lipophilic and hydrophilic type of statin (aHR 0.42, 95% CI 0.20–0.89). The risk was lower for patients receiving a cumulative defined daily dose (cDDD) of > 430 (aHR 0.41, 95% CI 0.19–0.86). This study showed that there is little association between statin use and second cancer risk in breast cancer patients.

Acinetobacter baumannii emerged as one of the most important pathogens that causes nosocomial infections due to its increased multidrug resistance. Identifying capsular epidemiology in A. baumannii can aid in the development of effective treatments and preventive measures against this emerging pathogen. Here we established a wzc-based method, and combined it with wzy-PCR to determine capsular types of A. baumannii causing nosocomial bacteraemia collected at two medical centres in Taiwan from 2015 to 2017. Among the 237 patients with A. baumannii bacteraemia, 98 (41.4%) isolates were resistant to carbapenems. Four prevalent capsular types (KL2, KL10, KL22, and KL52) accounted for 84.7% of carbapenem-resistant A. baumannii (CRAB) and 12.2% of non-CRAB. The rate of pneumonia, intensive care unit admission, APACHE II score, and Pitt bacteraemia score were higher in patients with KL2/10/22/52 infection than in those with non-KL2/10/22/52 infection. Patients with KL2/10/22/52 infection and patients with CRAB infection have a higher cumulative incidence of attributable and all-cause in-hospital 30-day mortality. On multivariate analysis, appropriate empirical antimicrobial therapy within 24 h was associated with a lower risk of 30-day attributable mortality in the KL2/10/22/52 isolates (odds ratio = 0.19, 95% CI: 0.06–0.66, p = 0.008) but not in non-KL2/10/22/52 isolates. Early recognition of carbapenem resistance-associated capsular types may help clinicians to promptly implement appropriate antimicrobial therapy for improving the outcomes in patients with CRAB bacteraemia.

Obesity has been associated with cognition in observational studies; however, whether its effect is confounding, reverse causality, or causal remains inconclusive. Using two-sample Mendelian randomization (MR) analyses, we investigated the causality of overall obesity, measured by BMI, and abdominal adiposity, measured by waist–hip ratio adjusted for BMI (WHRadjBMI), on cognition. Using summary data from the GIANT consortium, COGENT consortium, and UK Biobank of European ancestry, there was no causal effect of BMI on cognition performance (beta[95% CI]=-0.04[-0.12,0.04], p-value=0.35); however, a 1-SD increase in WHRadjBMI was associated with 0.07 standardized decrease in cognition performance (beta[95% CI]=-0.07[-0.12,-0.02], p=0.006). Using raw data from the Taiwan Biobank of Asian ancestry, there was no causal effect of BMI on cognitive aging (beta[95% CI]=0.00[-0.09,0.09], p-value=0.95); however, a 1-SD increase in WHRadjBMI was associated with a 0.17 standardized decrease in cognitive aging (beta[95% CI]=-0.17[-0.30,-0.03], p=0.02). This trans-ethnic MR study reveals that abdominal adiposity impairs cognition.

Dissemination of multidrug-resistant, particularly tigecycline-resistant, Acinetobacter baumannii is of critical importance, as tigecycline is considered a last-line antibiotic. Acquisition of tet(X), a tigecycline-inactivating enzyme mostly found in strains of animal origin, imparts tigecycline resistance to A. baumannii. Herein, we investigated the presence of tet(X) variants among 228 tigecycline-non-susceptible A. baumannii isolates from patients at a Taiwanese hospital via polymerase chain reaction using a newly designed universal primer pair. Seven strains (3%) carrying tet(X)-like genes were subjected to whole genome sequencing, revealing high DNA identity. Phylogenetic analysis based on the PFGE profile clustered the seven strains in a clade, which were thus considered outbreak strains. These strains, which were found to co-harbor the chromosome-encoded tet(X6) and the plasmid-encoded blaOXA-72 genes, showed a distinct genotype with an uncommon sequence type (Oxford ST793/Pasteur ST723) and a new capsular type (KL129). In conclusion, we identified an outbreak clone co-carrying tet(X6) and blaOXA-72 among a group of clinical A. baumannii isolates in Taiwan. To the best of our knowledge, this is the first description of tet(X6) in humans and the first report of a tet(X)-like gene in Taiwan. These findings identify the risk for the spread of tet(X6)-carrying tigecycline- and carbapenem-resistant A. baumannii in human healthcare settings.

BackgroundStreptococcus pneumoniaeis a common cause of post-influenza secondary bacterial infection, which results in excessive morbidity and mortality. Although 13-valent pneumococcal conjugate vaccine (PCV13) vaccination programs have decreased the incidence of pneumococcal pneumonia, PCV13 failed to prevent serotype 3 pneumococcal disease as effectively as other vaccine serotypes. We aimed to investigate the mechanisms underlying the co-pathogenesis of influenza virus and serotype 3 pneumococci.MethodsWe carried out a genome-wide screening of a serotype 3S. pneumoniaetransposon insertion mutant library in a mouse model of coinfection with influenza A virus (IAV) to identify the bacterial factors required for this synergism.ResultsDirect, high-throughput sequencing of transposon insertion sites identified 24 genes required for both coinfection and bacterial infection alone. Targeted deletion of the putative aminotransferase (PA) gene decreased bacterial growth, which was restored by supplementation with methionine. The bacterial burden in a coinfection with thePAgene deletion mutant and IAV in the lung was lower than that in a coinfection with wild-type pneumococcus and IAV, but was significantly higher than that in an infection with thePAgene deletion mutant alone. These data suggest that IAV infection alters host metabolism to benefit pneumococcal fitness and confer higher susceptibility to pneumococcal infection. We further demonstrated that bacterial growth was increased by supplementation with methionine or IAV-infected mouse lung homogenates.ConclusionsThe data indicates that modulation of host metabolism during IAV infection may serve as a potential therapeutic intervention against secondary bacterial infections caused by serotype 3 pneumococci during IAV outbreaks in the future.

ContextComorbid depression in patients with diabetes deteriorates the prognosis. Antidepressants might attenuate the adverse effects of depression; however, they are associated with cardiometabolic adverse effects.ObjectiveThis study aimed to explore the association between antidepressant treatment and advanced diabetic complications and mortality among patients with depression and diabetes mellitus.MethodsWe conducted a nationwide retrospective cohort study of 36 276 patients with depression and newly treated diabetes mellitus using Taiwan’s universal health insurance database. Antidepressant treatment patterns within a 6-month window were classified into none, poor, partial, and regular use, and we accounted for time-dependent variables in the Cox proportional hazards regression analysis with adjustment for time-dependent comorbidity and concomitant use of medications. Different classes of antidepressants were compared. Macro- and microvascular complications, as well as all-cause mortality, were the main outcomes. Benzodiazepines were chosen as negative control exposure.ResultsCompared with poor use of antidepressants, regular use was associated with a 0.92-fold decreased risk of macrovascular complications and a 0.86-fold decreased risk of all-cause mortality but not associated with microvascular complications. Regular use of selective serotonin reuptake inhibitors was associated with a 0.83- and 0.75-fold decreased risk of macrovascular complications and all-cause mortality, respectively. Regular use of tricyclic or tetracyclic antidepressants was associated with a 0.78-fold decreased risk of all-cause mortality. Regular use of benzodiazepine showed no association with diabetic outcomes.ConclusionRegular antidepressant use was associated with lower risk of advanced diabetic complications compared with poor adherence. Clinicians should emphasize antidepressant treatment adherence among patients with depression and diabetes mellitus.

Acinetobacter baumannii is the main cause of nosocomial infections, and the treatment of such infections has become more difficult due to the emergence of carbapenem resistance. This study focused on major carbapenemase genes and explored the association between carbapenemase genes, sequence types (ST types), and capsular types (K types). A total of 98 carbapenem-resistant A. baumannii (CRAB) strains were collected from two hospitals, the Chang Gung Memorial Hospital-Lin Kou branch (LCGMH) in northern Taiwan and the CGMH-Kaohsiung branch (KCGMH) in southern Taiwan, from 2015–2017. Major carbapenemase genes of class A, B, and D β-lactamases were detected by polymerase chain reaction. All strains except 1 were positive for blaOXA-51-like, 76 strains (77.6%) carried blaOXA-23-like, and 25 strains (25.5%) carried blaOXA-24-like. The regional distribution showed that blaOXA-23-like was more predominant than blaOXA-24-like in both hospitals (85.3% and 60% in LCGMH and KCGMH, respectively); however, blaOXA-24-like displayed a much higher percentage in KCGMH (46.7%) than in LCGMH (16.2%). Oxford multilocus sequence typing and global optimal eBURST analysis were conducted for 59 strains. We revealed the association between blaOXA gene patterns, ST types, and K types and demonstrated that four major K types, KL2, KL10, KL22, and KL52, which were associated with specific ST types, were mainly clustered into clonal complexes CC208 and CC549 (a unique clonal complex found in Taiwan). These findings provide important information for monitoring the epidemiology and dissemination of this pathogen.

Summary Klebsiella pneumoniae is an important human pathogen causing opportunistic nosocomial and community‐acquired infections. A major public health concern regarding K. pneumoniae is the increasing incidence of multidrug‐resistant strains. Here, we isolated three novel Klebsiella bacteriophages, KN1‐1, KN3‐1 and KN4‐1, which infect KN1, KN3 and K56, and KN4 types respectively. We determined their genome sequences and conducted a comparative analysis that revealed a variable region containing capsule depolymerase‐encoding genes. Recombinant depolymerase proteins were produced, and their enzymatic activity and specificity were evaluated. We identified four capsule depolymerases in these phages that could only digest the capsule types of their respective hosts. Our results demonstrate that the activities of these capsule depolymerases were correlated with the host range of each phage; thus, the capsule depolymerases are host specificity determinants. By generating a capsule mutant, we demonstrate that capsule was essential for phage adsorption and infection. Further, capsule depolymerases can enhance bacterial susceptibility to serum killing. The discovery of these phages and depolymerases lays the foundation for the typing of KN1, KN3, KN4 and K56 Klebsiella and could be useful alternative therapeutics for the treatment of K. pneumoniae infections.

Cholesterol-α-glucosyltransferase (CGT) encoded by the type 1 capsular polysaccharide biosynthesis protein J (capJ) gene of Helicobacter pylori converts cellular cholesterol into cholesteryl glucosides. H. pylori infection induces autophagy that may increase bacterial survival in epithelial cells. However, the role of H. pylori CGT that exploits lipid rafts in interfering with autophagy for bacterial survival in macrophages has not been investigated. Here, we show that wild-type H. pylori carrying CGT modulates cholesterol to trigger autophagy and restrain autophagosome fusion with lysosomes, permitting a significantly higher bacterial burden in macrophages than that in a capJ-knockout (∆CapJ) mutant. Knockdown of autophagy-related protein 12 impairs autophagosome maturation and decreases the survival of internalised H. pylori in macrophages. These results demonstrate that CGT plays a crucial role in the manipulation of the autophagy process to impair macrophage clearance of H. pylori.

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.

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.

The genome of the multihost bacteriophage ΦK64-1, capable of infecting Klebsiella capsular types K1, K11, K21, K25, K30, K35, K64, and K69, as well as new capsular types KN4 and KN5, was analyzed and revealed that 11 genes ( S1-1 , S1-2 , S1-3 , S2-1 , S2-2 , S2-3 , S2-4 , S2-5 , S2-6 , S2-7 , and S2-8 ) encode proteins with amino acid sequence similarity to tail fibers/spikes or lyases. S2-5 previously was shown to encode a K64 capsule depolymerase (K64dep). Specific capsule-degrading activities of an additional eight putative capsule depolymerases (S2-4 against K1, S1-1 against K11, S1-3 against K21, S2-2 against K25, S2-6 against K30/K69, S2-3 against K35, S1-2 against KN4, and S2-1 against KN5) was demonstrated by expression and purification of the recombinant proteins. Consistent with the capsular type-specific depolymerization activity of these gene products, phage mutants of S1-2 , S2-2 , S2-3 , or S2-6 lost infectivity for KN4, K25, K35, or K30/K69, respectively, indicating that capsule depolymerase is crucial for infecting specific hosts. In conclusion, we identified nine functional capsule depolymerase-encoding genes in a bacteriophage and correlated activities of the gene products to all ten hosts of this phage, providing an example of type-specific host infection mechanisms in a multihost bacteriophage. IMPORTANCE We currently identified eight novel capsule depolymerases in a multihost Klebsiella bacteriophage and correlated the activities of the gene products to all hosts of this phage, providing an example of carriage of multiple depolymerases in a phage with a wide capsular type host spectrum. Moreover, we also established a recombineering system for modification of Klebsiella bacteriophage genomes and demonstrated the importance of capsule depolymerase for infecting specific hosts. Based on the powerful tool for modification of phage genome, further studies can be conducted to improve the understanding of mechanistic details of Klebsiella phage infection. Furthermore, the newly identified capsule depolymerases will be of great value for applications in capsular typing.

Statins, 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors, have been found to provide protective effects against several bacterial infectious diseases. Although the use of statins has been shown to enhance antimicrobial treated Helicobacter pylori eradication and reduce H. pylori-mediated inflammation, the mechanisms underlying these effects remain unclear. In this study, in vitro and ex vivo macrophage models were established to investigate the molecular pathways involved in statin-mediated inhibition of H. pylori-induced inflammation. Our study showed that statin treatment resulted in a dose-dependent decrease in intracellular H. pylori burden in both RAW264.7 macrophage cells and murine peritoneal exudate macrophages (PEMs). Furthermore, statin yielded enhanced early endosome maturation and subsequent activation of the autophagy pathway, which promotes lysosomal fusion resulting in degradation of sequestered bacteria, and in turn attenuates interleukin (IL)-1β production. These results indicate that statin not only reduces cellular cholesterol but also decreases the H. pylori burden in macrophages by promoting autophagy, consequently alleviating H. pylori-induced inflammation.

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.

The Gram-negative saprophyteBurkholderia pseudomalleiis 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 ofB. pseudomalleiand an attenuated strain of the sameB. pseudomalleiisolate, 6 genesBPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, andBURPS1710A_1419were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional geneBPSL3147that 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::KmandBURPS1710A_1419::Kmmutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model.BPSL2033::Kmdemonstrated weak statistical significance (P=0.049) at 8 hours after infection in macrophage infection study but this was not seen inBURPS1710A_1419::Km. Nevertheless, complemented strains of both genes were able to partially restore the gene defects in bothin vitroandin vivostudies, thus suggesting that they individually play a minor role in the virulence ofB. pseudomallei.

Background. Peptidoglycan-associated lipoprotein (Pal), murein lipoprotein (LppA), and outer membrane protein A (OmpA) are dominant outer membrane proteins (OMPs) that are released by gram-negative bacteria during sepsis. OMPs are implicated in the maintenance of cell envelope integrity. Here, we characterize the roles of these OMPs in pathogenesis during bacteremia caused by Klebsiella pneumoniae. Methods. pal-, lppA-, and ompA-deficient K. pneumoniae strains were constructed using an unmarked deletion method. Serum sensitivity, antiphagocytosis activity, outer membrane permeability, and sensitivity to anionic detergents and antimicrobial polypeptides were determined for these OMP gene deletion mutants. The ability of these OMP gene deletion mutants to induce immune responses was compared with that of the wild-type strain in a bacteremic mouse model. Results. Klebsiella pneumoniae strains deleted for pal or lppA exhibited reduced protection from serum killing and phagocytosis; perturbation to the outer membrane permeability barrier and hypersensitivity to bile salts and sodium dodecyl sulfate. The strain mutated for lppA had reduced ability to activate Toll-like receptor 4. Immunization of mice with the pal or lppA mutant provided protection against infection by the wild-type strain. Conclusions. Our findings indicate that K. pneumoniae Pal and LppA proteins are important in the maintenance of cell integrity, contribute to virulence, and could be used as attenuated vaccines.

Analysis of the genome of Klebsiella pneumoniae NTUH-K2044 strain revealed the presence of two clustered regularly interspaced short palindromic repeats (CRISPR) arrays separated with CRISPR-associated (cas) genes. Carbapenem-resistant K. pneumoniae isolates were observed to be less likely to have CRISPR-Cas than sensitive strains (5/85 vs. 22/132). Removal of the transcriptional repressor, H-NS, was shown to prevent the transformation of plasmids carrying a spacer and putative proto-spacer adjacent motif (PAM). The CRISPR-Cas system also decreased pUC-4K plasmid stability, resulting in plasmid loss from the bacteria with acquisition of new spacers. Analysis of the acquired proto-spacers in pUC-4K indicated that 5′-TTN-3′ was the preferred PAM in K. pneumoniae. Treatment of cells by imipenem induced hns expression, thereby decreasing cas3 expression and consequently repressed CRISPR-Cas activity resulted in increase of plasmid stability. In conclusion, NTUH-K2044 CRISPR-Cas contributes to decrease of plasmid transformation and stability. Through repression of CRISPR-Cas activity by induced H-NS, bacteria might be more able to acquire DNA to confront the challenge of imipenem.

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 Δ clpX and Δ 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 Δ clpX 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.

Encapsulated Klebsiella pneumoniae is the predominant causative agent of pyogenic liver abscess, an emerging infectious disease that often complicates metastatic meningitis or endophthalmitis. The capsular polysaccharide on K. pneumoniae surface was determined as the key to virulence. Although the regulation of capsular polysaccharide biosynthesis is largely unclear, it was found that protein-tyrosine kinases and phosphatases are involved. Therefore, the identification and characterization of such kinases, phosphatases, and their substrates would advance our knowledge of the underlying mechanism in capsule formation and could contribute to the development of new therapeutic strategies. Here, we analyzed the phosphoproteome of K. pneumoniae NTUH-K2044 with a shotgun approach and identified 117 unique phosphopeptides along with 93 in vivo phosphorylated sites corresponding to 81 proteins. Interestingly, three of the identified tyrosine phosphorylated proteins, namely protein-tyrosine kinase (Wzc), phosphomannomutase (ManB), and undecaprenyl-phosphate glycosyltransferase (WcaJ), were found to be distributed in the cps locus and thus were speculated to be involved in the converging signal transduction of capsule biosynthesis. Consequently, we decided to focus on the lesser studied ManB and WcaJ for mutation analysis. The capsular polysaccharides of WcaJ mutant (WcaJY5F) were dramatically reduced quantitatively, and the LD(50) increased by 200-fold in a mouse peritonitis model compared with the wild-type strain. However, the capsular polysaccharides of ManB mutant (ManBY26F) showed no difference in quantity, and the LD(50) increased by merely 6-fold in mice test. Our study provided a clear trend that WcaJ tyrosine phosphorylation can regulate the biosynthesis of capsular polysaccharides and result in the pathogenicity of K. pneumoniae NTUH-K2044.

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.

A CMY-2-producing capsular type K2 Klebsiella pneumoniae strain (TVGHKP93) with multidrug resistance was isolated from a recurrent liver abscess in a patient who also carried a CMY-2-producing Escherichia coli strain (TVGHEC01) in the stool. TVGHKP93 retained its high virulence compared with that of the isogenic strain (TVGHKP60) with wild-type resistance from the first liver abscess. Our conjugation experiment showed the successful transfer of the bla CMY-2 -carrying plasmid from TVGHEC01 into TVGHKP60. The transconjugant showed both high virulence and the multidrug-resistant phenotype, as did TVGHKP93.

Klebsiella pneumoniae is an important pathogen that causes hospital-acquired septicemia and is associated with the recent emergence of community-acquired pyogenic liver abscess (PLA). Clinical typing suggests that K. pneumoniae infections originate from the gastrointestinal reservoir. However, the underlying mechanism remains unknown. Here, we have sought to determine how K. pneumoniae penetrates the intestinal barrier. We identified that bacteremia and PLA clinical isolates adhered to and invaded intestinal epithelial cells. Internalization of K. pneumoniae in three different human colonic cell lines was visualized by confocal microscopy and three-dimensional (3D) imaging. Using a Transwell system, we demonstrated that these K. pneumoniae isolates translocated across a polarized Caco-2 monolayer. No disruptions of transepithelial electrical resistance and altered distribution of tight junction protein ZO-1 or occludin were observed. Therefore, K. pneumoniae appeared to penetrate the intestinal epithelium via a transcellular pathway. Using specific inhibitors, we characterized the host signaling pathways involved. Inhibition by cytochalasin D and nocodazole suggested that actin and microtubule cytoskeleton were both important for K. pneumoniae invasion. A Rho inhibitor, ML141, LY294002, and an Akt1/2 inhibitor diminished K. pneumoniae invasion in a dose-dependent manner, indicating that Rho family GTPases and phosphatidylinositol 3-kinase (PI3K)/Akt signaling were required. By a mouse model of gastrointestinal colonization, in vivo invasion of K. pneumoniae into colonic epithelial cells was demonstrated. Our results present evidence to describe a possible mechanism of gastrointestinal translocation for K. pneumoniae . Cell invasion by manipulating host machinery provides a pathway for gut-colonized K. pneumoniae cells to penetrate the intestinal barrier and access extraintestinal locations to cause disease.

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.

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

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.