Advances in an In Vitro Tuberculosis Infection Model Using Human Lung Organoids for Host-Directed Therapies.

The emergence of drug-resistant Mycobacterium tuberculosis (M.tb) has led to the development of novel anti-tuberculosis (anti-TB) drugs. Common methods for testing the efficacy of new drugs, including two-dimensional cell culture models or animal models, have several limitations. Therefore, an appropriate model representative of the human organism is required. Here, we developed an M.tb infection model using human lung organoids (hLOs) and demonstrated that M.tb H37Rv can infect lung epithelial cells and human macrophages (hMφs) in hLOs. This novel M.tb infection model can be cultured long-term and split several times while maintaining a similar number of M.tb H37Rv inside the hLOs. Anti-TB drugs reduced the intracellular survival of M.tb in hLOs. Notably, M.tb growth in hLOs was effectively suppressed at each passage by rifampicin and bedaquiline. Furthermore, a reduction in inflammatory cytokine production and intracellular survival of M.tb were observed upon knockdown of MFN2 and HERPUD1 (host-directed therapeutic targets for TB) in our M.tb H37Rv-infected hLO model. Thus, the incorporation of hMφs and M.tb into hLOs provides a powerful strategy for generating an M.tb infection model. This model can effectively reflect host-pathogen interactions and be utilized to test the efficacy of anti-TB drugs and host-directed therapies.

Correction to: M1 macrophage dependent-p53 regulates the intracellular survival of mycobacteria.

The original version of this article unfortunately contains an error in the acknowledgement section. The text "Brain Korea 21 PLUS Project for Medical Science, Chungnam National University" was omitted by mistake. The correct and complete acknowledgment is given below: Acknowledgments This work was supported by the research fund of Chungnam National University and the Brain Korea 21 PLUS Project for Medical Science, Chungnam National University. The funders had no role in study design, data collection and analysis decision to publish, or preparation of the manuscript.

M1 macrophage dependent-p53 regulates the intracellular survival of mycobacteria.

Tumor suppressor p53 is not only affects immune responses but also contributes to antibacterial activity. However, its bactericidal function during mycobacterial infection remains unclear. In this study, we found that the p53-deficient macrophages failed to control Mycobacterium tuberculosis (Mtb), manifested as a lower apoptotic cell death rate and enhanced intracellular survival. The expression levels of p53 during Mtb infection were stronger in M1 macrophages than in M2 macrophages. The TLR2/JNK signaling pathway plays an essential role in the modulation of M1 macrophage polarization upon Mtb infection. It facilitates p53-mediated apoptosis through the production of reactive oxygen species, nitric oxide and inflammatory cytokines in Mtb-infected M1 macrophages. In addition, nutlin-3 effectively abrogated the intracellular survival of mycobacteria in both TB patients and healthy controls after H37Ra infection for 24 h, indicating that the enhancement of p53 production effectively suppressed the intracellular survival of Mtb in hosts. These results suggest that p53 can be a new therapeutic target for TB therapy.

Reactive oxygen species-mediated endoplasmic reticulum stress response induces apoptosis of Mycobacterium avium-infected macrophages by activating regulated IRE1-dependent decay pathway.

Mycobacterium avium, a slow-growing nontuberculous mycobacterium, causes fever, diarrhoea, loss of appetite, and weight loss in immunocompromised people. We have proposed that endoplasmic reticulum (ER) stress-mediated apoptosis plays a critical role in removing intracellular mycobacteria. In the present study, we investigated the role of the regulated IRE1-dependent decay (RIDD) pathway in macrophages during M. avium infection based on its role in the regulation of gene expression. The inositol-requiring enzyme 1 (IRE1)/apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK) signalling pathway was activated in macrophages after infection with M. avium. The expression of RIDD-associated genes, such as Bloc1s1 and St3gal5, was decreased in M. avium-infected macrophages. Interestingly, M. avium-induced apoptosis was significantly suppressed by pretreatment with irestatin (inhibitor of IRE1α) and 4µ8c (RIDD blocker). Macrophages pretreated with N-acetyl cysteine (NAC) showed decreased levels of reactive oxygen species (ROS), IRE1α, and apoptosis after M. avium infection. The expression of Bloc1s1 and St3gal5 was increased in NAC-pretreated macrophages following infection with M. avium. Growth of M. avium was significantly increased in irestatin-, 4µ8c-, and NAC-treated macrophages compared with the control. The data indicate that the ROS-mediated ER stress response induces apoptosis of M. avium-infected macrophages by activating IRE1α-RIDD. Thus, activation of IRE1α suppresses the intracellular survival of M. avium in macrophages.

Sclerotic changes of cavernous hemangioma in the cirrhotic liver: long-term follow-up using dynamic contrast-enhanced computed tomography.

PURPOSE: To determine the intra- and extralesional factors that predict sclerotic degeneration of hepatic hemangiomas in the cirrhotic liver on long-term follow-up computed tomography (CT) examinations. MATERIALS AND METHODS: Fifty-seven hepatic hemangiomas (> 5 mm in diameter) in 41 cirrhotic patients, recruited over a 5-year period (January 2005-December 2009), were subjected to CT to determine which factors predict sclerotic contraction or degeneration in hemangiomas. Prior and follow-up CT examinations (from 2000 to 2018) were included to observe time-related changes. The patients' gender, age, cause of cirrhosis, progression of background liver cirrhosis, lesion size/location/contrast enhancement pattern, and serum aspartate transaminase to platelet ratio index were correlated with sclerotic changes of each lesion. RESULTS: According to the dynamic CT features, 36 of 57 (63%) hemangiomas were determined to have sclerotic changes during the follow-up period (1.1-14.4 years, median: 7.8 years), including 28 lesions (49%) reduced by ≥ 20% in diameter. In univariate analysis, age (p = 0.047) and morphological progression of background cirrhosis (p = 0.013) were significantly related to sclerotic change of hemangiomas. In the logistic regression analysis, only morphological progression of background liver cirrhosis independently predicted sclerotic change (odds ratio: 4.88, p = 0.007). With the exception of exophytic location free from size reduction (p = 0.023 in multivariate analysis), no other analyzed factors were significantly correlated with sclerotic changes. CONCLUSION: Overall, sclerotic changes of hepatic cavernous hemangioma followed the morphological progression of background liver cirrhosis, while exophytic lesions tended to be free of size reduction.

TNF-α-mediated ER stress causes elimination of Mycobacterium fortuitum reservoirs by macrophage apoptosis.

Mycobacterium fortuitum (MF), a rapidly growing nontuberculosis mycobacterium, is recognized as an important human pathogen. We investigated whether the endoplasmic reticulum (ER) stress response is associated with the apoptosis of MF-infected macrophages. The expression of ER molecular chaperones was significantly induced by MF infection. We found that MF-induced reactive oxygen species (ROS) generation plays a critical role in the induction of ER stress-mediated apoptosis. Excess TNF-α in the ER led to ER stress-mediated apoptosis during MF infection. The intracellular survival of MF was significantly increased by TNF-α knockdown compared with the control. This is the first report of MF-induced TNF-α as a cause of ER stress in macrophages. Furthermore, we found that TLR2-mediated ER stress response contributed to the elimination of intracellular MF in vivo. These results suggest that TNF-α-mediated ER stress during MF infection contributes to the suppression of intracellular survival of MF in macrophages. Our findings provide new insight into the importance of ER stress in mycobacterial infection.-Oh, S.-M., Lim, Y.-J., Choi, J.-A., Lee, J., Cho, S.-N., Go, D., Kim, S.-H., Song, C.-H. TNF-α-mediated ER stress causes elimination of Mycobacterium fortuitum reservoirs by macrophage apoptosis.

Toll-like receptor 9 ligands increase type I interferon induced B-cell activating factor expression in chronic rhinosinusitis with nasal polyposis.

B-cell activating factor (BAFF) has been proposed to play a crucial role in the pathogenesis of chronic rhinosinusitis with nasal polyp (CRSwNP). The aim of this study was to evaluate the role of toll-like receptor (TLR) 9-mediated BAFF activation on the pathogenesis of CRSwNP. NP and uncinate tissue (UT) were obtained from patients with CRSwNP or CRS without NP, and control subjects. The expression of TLR9, high mobility group box-1 protein (HMGB1), type I interferon (IFN), BAFF, and anti-double stranded DNA (dsDNA) antibody were examined in the tissues and the cultured dispersed NP cells (DNPCs). The expression of TLR9, HMGB1, type I IFN, BAFF, and anti-dsDNA antibody were elevated in NP tissue compared to the UTs. Exposure to TLR9 agonist increased the type I IFN expression in vitro, which further increased BAFF production. In conclusion, we provided a novel therapeutic potential of TLR9 agonist in CRSwNP.

Mycobacterium tuberculosis 38-kDa antigen induces endoplasmic reticulum stress-mediated apoptosis via toll-like receptor 2/4.

Endoplasmic reticulum (ER) stress responses play critical roles in the pathogenesis of tuberculosis. To investigate the regulatory role of the ER stress response in 38-kDa antigen-induced apoptosis, we examined the relationship between the ER stress response and apoptosis in bone marrow-derived macrophages (BMDMs) stimulated with Mycobacterium tuberculosis antigen (38-kDa Ag). The expression of ER molecular chaperones, including C/EBP homologous protein (CHOP), glucose-regulated protein (Bip) and phosphorylated alpha subunit of eukaryotic initiation factor 2, was induced in BMDMs stimulated with the 38-kDa Ag. Interestingly, 38-kDa Ag-stimulation induced apoptosis via activation of caspase-12, -9 and -3. However, 38-kDa Ag-induced apoptosis was significantly reduced in TLR2- and TLR4-deficient macrophages. Because toll-like receptors (TLRs) initiate the activation of mitogen-activated protein kinase (MAPK) signaling cascades, we evaluated the effect of MAPK activation on ER stress. The 38-kDa Ag activated Jun N-terminal kinase, extracellular signal-regulated kinase and p38 phosphorylation. MAPK signaling induced the secretion of proinflammatory cytokines such as MCP-1, TNF-α and IL-6. The 38-kDa Ag-induced MCP-1 was especially associated with the induction of MCP-1-induced protein (MCPIP), which increased the generation of reactive oxygen species (ROS) and ER stress. To investigate the role of MCPIP in ROS-induced ER stress by 38-kDa Ag stimulation, we transfected MCPIP siRNA into RAW264.7 cells before 38-kDa Ag stimulation, and measured the generation of ROS and expression of ER molecular chaperones. ROS production and CHOP expression were decreased by the silencing of MCPIP induction. Our results demonstrate that the expression of MCPIP by 38-kDa Ag stimulation is increased through a TLR-MAPK-dependent signaling pathway, and leads to ER stress-induced apoptosis. In conclusion, MCPIP is important for host defense mechanisms in mycobacterial pathogenesis.

Evaluation of four methods of assigning species and genus to medically important bacteria using 16S rRNA gene sequence analysis.

The four methods for assigning bacterial species are the Clinical and Laboratory Standards Institute (CLSI), modified CLSI (mCLSI), phylogenetic analysis (PA) and closest match (CM) methods, these are used to identify the genus and species using 16S rRNA gene sequence results. In this study, the results of identification by these four methods of 37 aerobic reference strains, 30 anaerobic reference strains, 15 Acinetobacter reference strains and 167 Acinetobacter clinical strains were compared. The rates of accurate identification to the species level using the CLSI, mCLSI, PA and CM methods were as follows: 24.3, 86.5, 86.5 and 89.2%, respectively, for the 37 aerobic reference strains; 73.3%, 96.7%, 90.0% and 93.3%, respectively, for the 30 anaerobic reference strains; 40.0%, 93.3%, 100% and 93.3%, respectively, for the 15 Acinetobacter reference strains; and 53.9%, 90.4%, 95.8% and 90.4%, respectively, for the 167 Acinetobacter clinical strains. The rates of accurate identification to the genus level using the CLSI, mCLSI, PA, and CM methods were as follows: 91.9%, 91.9%, 94.6% and 91.9%, respectively, for the 37 aerobic reference strains; 100%, 100%, 100% and 100%, respectively, for all of the 30 anaerobic reference strains, 15 Acinetobacter reference strains and the 167 Acinetobacter clinical strains. The mCLSI is the most practical and pragmatic method for identification of species based on 16S rRNA sequences for hospital, research or industry laboratories because it performs well and involves a simple procedure.

Mycobacterium kansasii-induced death of murine macrophages involves endoplasmic reticulum stress responses mediated by reactive oxygen species generation or calpain activation.

Although pathogenic mechanisms of tuberculosis have been extensively studied, little is known about the pathogenic mechanisms of Mycobacterium kansasii. In this work the influence of virulence and ER-stress mediated apoptosis of macrophages during two different strains of M. kansasii infection was investigated. We show that M. kansasii infection is associated with ER stress-mediated apoptosis in the murine macrophage cell line RAW 264.7. Infection of RAW 264.7 cells in vitro with apoptosis-inducing a clinical isolate of M. kansasii SM-1 (SM-1) resulted in strong induction of ER stress responses compared with M. kansasii type strain (ATCC 12478)-infected RAW 264.7 cells. Interestingly, inhibition of calpain prevented the induction of CHOP and Bip in ATCC 12478-infected RAW 264.7 cells but not in RAW 264.7 cells infected with SM-1. In contrast, reactive oxygen species (ROS) were significantly increased only in RAW 264.7 cells infected with SM-1. We propose that ROS generation is important for triggering ER stress-mediated apoptosis during SM-1 infection, whereas ATCC 12478-induced, ER stress-mediated apoptosis is associated with calpain activation. Our results demonstrate that the ER stress pathway plays important roles in the pathogenesis of M. kansasii infections, and that different strains of M. kansasii induce different patterns of ER stress-mediated apoptosis.

Impaired mitophagy induces antimicrobial responses in macrophages infected with Mycobacterium tuberculosis.

BACKGROUND: Mitophagy, mitochondrial selective autophagy, plays a pivotal role in the maintenance of cellular homeostasis in response to cellular stress. However, the role of mitophagy in macrophages during infection has not been elucidated. To determine whether mitophagy regulates intracellular pathogen survival, macrophages were infected with Mycobacterium tuberculosis (Mtb), an intracellular bacterium. RESULTS: We showed that Mtb-infected macrophages induced mitophagy through BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) activation. In contrast, BNIP3-deficient macrophages failed to induce mitophagy, resulting in reduced mitochondrial membrane potential in response to Mtb infection. Moreover, the accumulation of damaged mitochondria due to BNIP3 deficiency generated higher levels of mitochondrial reactive oxygen species (mROS) compared to the control, suppressing the intracellular survival of Mtb. We observed that siBNIP3 suppressed intracellular Mtb in mice lungs. CONCLUSION: We found that BNIP3 plays a critical role in the regulation of mitophagy during Mtb infection. The inhibition of mitophagy suppresses Mtb growth in macrophages through increased mROS production. Therefore, BNIP3 might be a novel therapeutic target for tuberculosis treatment.

Herp regulates intracellular survival of Mycobacterium tuberculosis H37Ra in macrophages by regulating reactive oxygen species-mediated autophagy.

IMPORTANCE: Several studies have suggested that endoplasmic reticulum (ER) stress is important in the pathogenesis of infectious diseases; however, the precise function of ER stress regulation and the role of Herp as a regulator in Mtb H37Ra-induced ER stress remain elusive. Therefore, our study investigated ER stress and autophagy associated with Herp expression in Mycobacterium tuberculosis-infected macrophages to determine the role of Herp in the pathogenesis of tuberculosis.

The association of oxidative stress with central obesity in obstructive sleep apnea.

PURPOSE: The purpose of this paper is to evaluate concentrations of multiple oxidative stress and antioxidant status markers in patients with obstructive sleep apnea (OSA) and normal controls of comparable obesity. METHODS: A total of 73 male subjects, recruited from a sleep clinic or advertisements, were enrolled in this study. All subjects underwent overnight polysomnography. The subjects were divided into normal control (n = 20), mild to moderate OSA (n = 31), and severe OSA (n = 22) with no difference in obesity. Blood was withdrawn from subjects, and markers of oxidative stress and antioxidant status, and parameters of glucose metabolism were assessed. RESULTS: There was no significant difference in either oxidative stress or antioxidant status markers among the three groups. There was no significant correlation between the oxidative stress markers and the OSA variables. However, there were correlations between waist-to-hip ratio (WHR)-oxidized low-density lipoprotein cholesterol (oxLDL) (r = 0.424), WHR-glutathione peroxidase (GPX) (r = 0.318), WHR-total antioxidant status (TAS) (r = -0.317), and WHR-superoxide dismutase (SOD) (r = -0.338). In multiple regression analysis, WHR was a significant independent variable of oxLDL, GPX, TAS, and SOD. CONCLUSIONS: The oxidative stress in OSA was related to central obesity rather than intermittent hypoxia or respiratory disturbances. To control cardiovascular complications in OSA, weight reduction should be a component in the treatment strategy.

Pulmonary nodular lymphoid hyperplasia presenting as multifocal subsolid nodules: A case report and literature review.

Pulmonary nodular lymphoid hyperplasia (PNLH) is a rare, benign lymphoproliferative disease, which is characterized by nonclonal lymphoproliferation. PNLH is usually asymptomatic and usually detected incidentally on imaging studies. Common imaging findings include a solitary nodule, multiple nodules, or focal consolidation. Atypically, PNLH may present with persistent subsolid nodules, mimicking adenocarcinoma. Here, we report a rare case of PNLH presenting as multifocal subsolid nodules in both lower lobes. During follow-up, persistency and growth of the subsolid nodules suggested the possibility of malignancy. Wedge resection was performed bilaterally, and PNLH was confirmed on pathological examination.

Lipocalin 2 regulates expression of MHC class I molecules in Mycobacterium tuberculosis-infected dendritic cells via ROS production.

BACKGROUND: Iron has important roles as an essential nutrient for all life forms and as an effector of the host defense mechanism against pathogenic infection. Lipocalin 2 (LCN2), an innate immune protein, plays a crucial role in iron transport and inflammation. In the present study, we examined the role of LCN2 in immune cells during Mycobacterium tuberculosis (Mtb) infection. RESULTS: We found that infection with Mtb H37Ra induced LCN2 production in bone marrow-derived dendritic cells (BMDCs). Notably, expression of MHC class I molecules was significantly reduced in LCN2-/- BMDCs during Mtb infection. The reduced expression of MHC class I molecules was associated with the formation of a peptide loading complex through LCN2-mediated reactive oxygen species production. The reduced expression of MHC class I molecules affected CD8+ T-cell proliferation in LCN2-/- mice infected with Mtb. The difference in the population of CD8+ effector T cells might affect the survival of intracellular Mtb. We also found a reduction of the inflammation response, including serum inflammatory cytokines and lung inflammation in LCN2-/- mice, compared with wild-type mice, during Mtb infection. CONCLUSIONS: These data suggest that LCN2-mediated reactive oxygen species affects expression of MHC class I molecules in BMDCs, leading to lower levels of CD8+ effector T-cell proliferation during mycobacterial infection.

Ang II-Induced Hypertension Exacerbates the Pathogenesis of Tuberculosis.

It has been known that infection plays a role in the development of hypertension. However, the role of hypertension in the progression of infectious diseases remain unknown. Many countries with high rates of hypertension show geographical overlaps with those showing high incidence rates of tuberculosis (TB). To explore the role of hypertension in tuberculosis, we compared the effects of hypertension during mycobacterial infection, we infected both hypertensive Angiotensin II (Ang II) and control mice with Mycobacterium tuberculosis (Mtb) strain H37Ra by intratracheal injection. Ang II-induced hypertension promotes cell death through both apoptosis and necrosis in Mtb H37Ra infected mouse lungs. Interestingly, we found that lipid accumulation in pulmonary tissues was significantly increased in the hypertension group compared to the normal controls. Ang II-induced hypertension increases the formation of foamy macrophages during Mtb infection and it leads to cell death. Moreover, the hypertension group showed more severe granuloma formation and fibrotic lesions in comparison with the control group. Finally, we observed that the total number of mycobacteria was increased in the lungs in the hypertension group compared to the normal controls. Taken together, these results suggest that hypertension increases intracellular survival of Mtb through formation of foamy macrophages, resulting in severe pathogenesis of TB.

Development of a Two Triplex Real-Time Polymerase Chain Reaction for Rapid Detection of Six Carbapenemase Genes in Enterobacteriaceae.

OBJECTIVES: Carbapenem resistance is a serious clinical and public health threat. Carbapenemase can confer carbapenem resistance, and most carbapenemase genes are plasmid encoded so resistance can easily spread. In this study, we aimed to develop a novel system based on the TaqMan platform for the rapid detection of 6 clinically prevalent carbapenemase genes: Klebsiella pneumoniae carbapenemase, New Delhi metallo-ß-lactamase, oxacillinase, imipenem-hydrolyzing, Verona integron-encoded metallo-ß-lactamase, and Guiana extended-spectrum ß-lactamase. METHODS: The triplex assay was verified by testing genomic DNA of 6 carbapenemase-producing Klebsiella pneumoniae. It was validated with a blinded panel of 310 Enterobacteriaceae isolates, including 225 carbapenemase-producers and 85 non-producers, by direct colony triplex real-time polymerase chain reaction (PCR). The real-time PCR was performed using the ABI 7500 fast instrument (Applied Biosystems, CA, USA) and specific primers for each carbapenemase target were designed to include modified peptide-nucleic acid oligonucleotides. RESULTS: No amplification was detected among the negative samples. The result showed 100% concordance with the genotypes previously identified. The entire assay, including DNA extraction and real-time PCR, was completed within 2 hours. CONCLUSION: The newly developed triplex real-time PCR assay was useful for the rapid, accurate and simultaneous detection of 6 carbapenemase genes in Enterobacteriaceae, suggesting its potential to allow an early decision on the appropriate treatment, management, and prevention of the spread of resistant infections in hospitals.

Insights Into the Role of Endoplasmic Reticulum Stress in Infectious Diseases.

The endoplasmic reticulum (ER) is the major organelle in the cell for protein folding and plays an important role in cellular functions. The unfolded protein response (UPR) is activated in response to misfolded or unfolded protein accumulation in the ER. However, the UPR successfully alleviates the ER stress. If UPR fails to restore ER homeostasis, apoptosis is induced. ER stress plays an important role in innate immune signaling in response to microorganisms. Dysregulation of UPR signaling contributes to the pathogenesis of a variety of infectious diseases. In this review, we summarize the contribution of ER stress to the innate immune response to invading microorganisms and its role in the pathogenesis of infectious diseases.

Mitofusin 2-Deficiency Suppresses Mycobacterium tuberculosis Survival in Macrophages.

Apoptosis is an important host defense mechanism against mycobacterial infection. However, the molecular mechanisms regulating apoptosis during mycobacterial infection are not well known. Recent reports suggest that bacterial infection regulates mitochondrial fusion and fission in various ways. Here, we investigated the role of mitochondria in Mycobacterium tuberculosis (Mtb)-infected macrophages. Mtb H37Rv (Rv) infection induced mitofusin 2 (MFN2) degradation, leading to mitochondrial fission. Interestingly, Mtb H37Ra (Ra) infection induced significantly greater mitochondrial fragmentation than Rv infection. Mtb-mediated Parkin, an E3 ubiquitin ligase, contributed to the degradation of MFN2. To evaluate the role of endoplasmic reticulum stress in the production of Parkin during Mtb infection, we analyzed Parkin production in 4-phenylbutyric acid (4-PBA)-pretreated macrophages. Pretreatment with 4-PBA reduced Parkin production in Mtb-infected macrophages. In contrast, the level of MFN2 production recovered to a level similar to that of the unstimulated control. In addition, Ra-infected macrophages had reduced mitochondrial membrane potential (MMP) compared to those infected with Rv. Interestingly, intracellular survival of mycobacteria was decreased in siMFN2-transfected macrophages; in contrast, overexpression of MFN2 in macrophages increased Mtb growth compared with the control.

MCR1 and KPC2 Co-producing Klebsiella pneumoniae Bacteremia: First Case in Korea.

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) has been disseminating nationwide due to clonal spread and is taking a serious action at the national level in Korea. The mobilized colistin resistance (MCR1) gene confers plasmid-mediated resistance to colistin and is known to be capable of horizontal transfer between different strains of a bacterial species. We have experienced a fatal case of the patient who developed MCR1-possessing, ST307/Tn4401a[blaKPC2] K. pneumonia bacteremia in the community of non-capital region after being diagnosed as pancreatic cancer with multiple liver metastases and treated in the capital region. The ST307/Tn4401a[blaKPC2] K. pneumonia was the most commonly disseminated clone in Korea. Our strain is the first MCR1 and KPC2 co-producing K. pneumonia in Korea and our case is the critical example that the multi-drug resistant clone can cause inter-regional spread and the community-onset fatal infections. Fortunately, our patient was admitted to the intensive care unit on the day of visit, and the contact precaution was well maintained throughout and KPC-KP was not spread to other patients. The high risk patients for KPC-KP need to be screened actively, detected rapidly and preemptively isolated to prevent outbreak of KPC-KP. Inter-facility communications are essential and the nationwide epidemiologic data of KPC-KP should be analyzed and reported regularly to prevent spread of KPC-KP. The prompt identification of species and antimicrobial susceptibilities for successful treatment against KPC-KP should be emphasized as well.