Molecular characterization and antibiotic resistance of Staphylococcus aureus strains isolated from patients with diarrhea in Korea between the years 2007 and 2022.

To investigate the molecular characteristics and antibiotic resistance of Staphylococcus aureus isolates from patients with diarrhea in Korea, 327 S. aureus strains were collected between 2007 and 2022. The presence of staphylococcal enterotoxin (SE) and toxic shock syndrome toxin-1 (TSST-1) genes in S. aureus isolates was determined by PCR. The highest expression of the TSST-1 gene was found in the GIMNO type (43.1% of GIMNO type). GIMNO type (Type I) refers to each staphylococcal enterotoxin (SE) gene gene (initials of genes): G = seg; I = sei; M = selm; N = seln; O = selo. Moreover, Type I isolates showed a significantly higher resistance to most antibiotics. A total of 195 GIMNO-type S. aureus strains were analyzed using multilocus sequence typing (MLST), and 18 unique sequence types (STs) were identified. The most frequent sequence type was ST72 (36.9%), followed by ST5 (22.1%) and ST30 (16.9%). Interestingly, ST72 strains showed a higher prevalence of MRSA than the other STs. In conclusion, our results were the first reported for S. aureus strains in Korea, which significantly expanded S. aureus genotype information for the surveillance of pathogenic S. aureus and may provide important epidemiological information to resolve several infectious diseases caused by S. aureus. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01478-9.

A novel Mycobacterium Tuberculosis antigen, MTB48 enhances inflammatory response in LPS-induced RAW264.7 macrophage immune cells.

Mtb (Mycobacterium tuberculosis) is a pathogenic bacterium that causes tuberculosis infection (TB). Mtb-secreted proteins have recently been investigated as virulence factors, as well as therapeutic and vaccine possibilities. The early-secreted antigen target MTB48 is one of these proteins that has been explored as a cocktail antigen in the serodiagnosis of active tuberculosis. However, there exists no information about the function or control of MTB48's inflammatory activity in macrophages at the site of inflammation. As a result, the goal of this research was to figure out what processes are involved in MTB48's function. MTB48 stimulated inflammation in LPS induced macrophages at both the protein and mRNA levels, which was interesting. MTB48 aided LPS induced IB phosphorylation and NF-κB translocation. MTB48 also led to the phosphorylation of MAPK signaling protein. These findings imply that MTB48 can enhance inflammatory activity via NF-κB and MAPK signaling by upregulating COX-2, iNOS, NO and PGE2. Many tuberculosis antigens have been tested for the development of rapid serological diagnosis. The results of this study suggest that MTB48 is a very high conservative antigen and is a major factor causing inflammatory reactions, suggesting that it can help control and diagnose tuberculosis.

GM1a ganglioside-binding domain peptide inhibits host adhesion and inflammatory response of enterotoxigenic Escherichia coli heat-labile enterotoxin-B in HCT-8 cells.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of illness and death but has no effective therapy. The heat-labile enterotoxin LT is a significant virulence factor produced by ETEC. The heat-labile enterotoxin-B (LT-B) subunit may enter host cells by binding to monosialotetrahexosylganglioside-a (GM1a), a monosialoganglioside found on the plasma membrane surface of animal epithelial cells. This research was conducted to develop conformationally comparable peptides to the carbohydrate epitope of GM1a for the treatment of ETEC. We used the LT-B subunit to select LT-B-binding peptides that structurally resemble GM1a. The ganglioside microarray and docking simulations were used to identify three GM1a ganglioside-binding domain (GBD) peptides based on LT-B recognition. Peptides had an inhibiting effect on the binding of LT-B to GM1a. The binding capacity, functional inhibitory activity, and in vitro effects of the GBD peptides were evaluated using HCT-8 cells, a human intestinal epithelial cell line, to evaluate the feasibility of deploying GBD peptides to combat bacterial infections. KILSYTESMAGKREMVIIT was the most efficient peptide in inhibiting cellular absorption of LT-B in cells. Our findings offer compelling evidence that GM1a GBD-like peptides might act as new therapeutics to inhibit LT-B binding to epithelial cells and avoid the subsequent physiological consequences of LT.

Up-regulation of inflammatory reactions by MPT32, a secreted protein of Mycobacterium tuberculosis in RAW264.7 macrophages.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and is still one of the global health burdens. The occurrence of various cases and multidrug resistance confirm that TB has not been completely conquered. For these reasons, the present research has been conducted to explore TB vaccine and drug candidate possibility using Mtb-secreted proteins. Among these proteins, MPT32 is known to have antigenicity and immunogenicity. There has not been a report on the host immune responses and regulation in macrophage cells. The present study was conducted with MPT32 in RAW 264.7 murine macrophage cells that control immune responses by sensing pathogen invasion and environmental change. We have found that MPT32 could activate lipopolysaccharide (LPS)-induced gene expression of metalloproteinase-9 (MMP-9) and inflammation in RAW 264.7 cells. After treating cells with MPT32, the increase in pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß (IL-1ß) and IL-6, was observed. In addition, activated macrophages expressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) to generate various inflammatory mediator molecules, such as nitric oxide (NO). The increase in iNOS and COX-2 levels, which are up-regulators of MMP-9 expression, was also confirmed. The biochemical events are involved in the downstream of activated MAPK signaling and translocation of NF-κ B transcription factor. The present results prove the immunomodulatory effect of MPT32 in the RAW 264.7 murine macrophage cells. it claims the possibility of a TB vaccination and drug candidate using MPT32, contributing to the prevention of TB.

Production of monoclonal antibody of heat-labile toxin A subunit to identify enterotoxigenic Escherichia coli by epitope mapping using synthetic peptides.

Background: Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea through two enterotoxins, a heat-labile toxin and a heat-stable toxin. These toxins alter the cellular signaling pathways, ultimately triggering an increase in chloride secretion and watery diarrhea. Objective: For the development of an ETEC vaccine, we attempted to construct a peptide-specific monoclonal antibody library against heat-labile enterotoxin A subunit (LT-A) by epitope mapping using synthetic peptides. Methods: Sera produced by five mice immunized with recombinant LT-A protein were examined for specific recognition with synthetic 15-mer and 34-mer peptides of LT-A proteins using enzyme-linked immunosorbent assay. The analysis revealed that the synthetic peptides number 8, 16, 24, 33, 36, 38, and 39 reacted with an anti-LT-A polyclonal antibody. For the possible prediction of LT-A epitopes, each full-length protein sequence was subjected to BCPreds analysis and three-dimensional protein structure analysis. The data showed that three peptides (synthetic peptide numbers: 33, 36, and 38-39) have identical antigenic specificities with LT-A protein, suggesting the usefulness of these linear peptide epitopes. Results: Based on these peptides, we produced monoclonal antibodies to improve the specificity of LT-A detection. Monoclonal antibodies produced from two peptides (numbers 33 and 36) showed affinity for an LT-A recombinant antigen. Moreover, peptide epitope prediction analysis showed that the sites of the three peptides were identical to those exhibiting actual antigenicity. Also, it was confirmed that the amino acid sequence that actually showed antigenicity was included in the peptide predicted only by ETEC-LT-A-33. Also, the specificity of the antibody for ETEC-LT-A-33 was validated using bacterial cells, and the neutralizing effect of the antibody was determined by assessing cytokine release in infected HCT-8 cells. Conclusion: The monoclonal antibodies produced in this study are useful toolsfor vaccine production against ETEC and can be used to identify peptide antigencandidates.

Glycan-Adhering Lectins and Experimental Evaluation of a Lectin FimH Inhibitor in Enterohemorrhagic Escherichia coli (EHEC) O157:H7 Strain EDL933.

In this study, we tried to develop a FimH inhibitor that inhibits adhesion of enterohemorrhagic Escherichia coli (EHEC) on the epithelium of human intestine during the initial stage of infections. Using a T7 phage display method with a reference strain, EHEC EDL933, FimH was selected as an adherent lectin to GM1a and Gb3 glycans. In order to detect the ligand binding domain (LBD) of FimH, we used a docking simulation and found three binding site sequences of FimH, i.e., P1, P2, and P3. Among Gb3 mimic peptides, P2 was found to have the strongest binding strength. Moreover, in vitro treatment with peptide P2 inhibited binding activity in a concentration-dependent manner. Furthermore, we conducted confirmation experiments through several strains isolated from patients in Korea, EHEC NCCP15736, NCCP15737, and NCCP15739. In addition, we analyzed the evolutionary characteristics of the predicted FimH lectin-like adhesins to construct a lectin-glycan interaction (LGI). We selected 70 recently differentiated strains from the phylogenetic tree of 2240 strains with Shiga toxin in their genome. We can infer EHEC strains dynamically evolved but FimH was conserved during the evolution time according to the phylogenetic tree. Furthermore, FimH could be a reliable candidate of drug target in terms of evolution. We examined how pathogen lectins interact with host glycans early in infection in EDL933 as well as several field strains and confirmed that glycan-like peptides worked as an initial infection inhibitor.

Systemic Lectin-Glycan Interaction of Pathogenic Enteric Bacteria in the Gastrointestinal Tract.

Microorganisms, such as bacteria, viruses, and fungi, and host cells, such as plants and animals, have carbohydrate chains and lectins that reciprocally recognize one another. In hosts, the defense system is activated upon non-self-pattern recognition of microbial pathogen-associated molecular patterns. These are present in Gram-negative and Gram-positive bacteria and fungi. Glycan-based PAMPs are bound to a class of lectins that are widely distributed among eukaryotes. The first step of bacterial infection in humans is the adhesion of the pathogen's lectin-like proteins to the outer membrane surfaces of host cells, which are composed of glycans. Microbes and hosts binding to each other specifically is of critical importance. The adhesion factors used between pathogens and hosts remain unknown; therefore, research is needed to identify these factors to prevent intestinal infection or treat it in its early stages. This review aims to present a vision for the prevention and treatment of infectious diseases by identifying the role of the host glycans in the immune response against pathogenic intestinal bacteria through studies on the lectin-glycan interaction.

Evaluation of Microbiological Contamination of Dummies Used in Cardiopulmonary Resuscitation in Korea.

OBJECTIVES: In order to prevent infections through dummies used during Cardiopulmonary Resuscitation (CPR) training, we analyzed the microbiological contamination on dummies used in CPR institutions. METHODS: A total of 31 dummy samples were collected from 13 different institutions in Korea, and were evaluated for the number of contaminating bacteria and fungi on the surface. PCR and biochemical tests were performed to identify pathogenic bacteria and fungi, including Methicillin-Resistant Staphylococcus aureus (MRSA). Moreover, we further assessed the survival rate of microorganisms on the surface of the dummies. RESULTS: We assessed the total number of microorganisms on the surface to be 77,752CFU/cm2 (±50,047CFU), which is up to 188 times higher than the required surface contamination level. Grampositive cocci such as Micrococcus spp. and Staphylococcus spp. accounted for the highest proportion (55.3%). Especially, we detected three MRSA strains. Considering the isolated fungi and yeast, Aspergillus spp. and Candidia spp. accounted for the highest proportion. Assessing the contamination level simulation and survival rate on the humanoid surface showed that within two weeks of training, the level of contamination on the dummy's surface exceeded the standard, and artificially contaminated pathogenic strains on the surface of the dummy survived for at least 40 days. CONCLUSION: To minimize the possibility of secondary infections during CPR training, there is a requirement for a standardized protocol for proper microbiological management of dummies.

e-Membranome: A Database for Genome-Wide Analysis of Escherichia coli Outer Membrane Proteins.

OBJECTIVE: Lectin-like adhesins of enteric bacterial pathogens such as Escherichia coli are an attractive target for vaccine or drug development. Here, we have developed e-Membranome as a database of genome-wide putative adhesins in Escherichia coli (E. coli). METHODS: The outer membrane adhesins were predicted from the annotated genes of Escherichia coli strains using the PSORTb program. Further analysis was performed using Interproscan and the String database. The candidate proteins can be investigated for homology modeling of the Three-Dimensional (3D) structure (I-TASSER version 5.1), epitope region (ABCpred), and the glycan array. RESULTS: e-Membranome is implemented using the Django (version 2.2.5) framework. The Web Application Server Apache Tomcat 6.0 is integrated into the platform on Ubuntu Linux (version 16.04). MySQL database (version 5.7) is used as a database engine. The information on homology model of the 3D structure, epitope region, and affinity information from the glycan array will be stored in the e- Membranome database. As a case study, we performed a genome-wide screening of outer membraneembedded proteins from the annotated genes of E. coli using the e-Membranome pipeline. CONCLUSION: This platform is expected to be a valuable resource for advancing research of outer membrane proteins for the construction of lectin-glycan interaction network of E. coli. In addition, the e- Membranome pipeline can be extended to other similar biological systems that need to address hostpathogen interactions.

Construction of a Lectin-Glycan Interaction Network from Enterohemorrhagic Escherichia coli Strains by Multi-omics Analysis.

Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis and hemolytic uremic syndrome. EHEC infection begins with bacterial adherence to the host intestine via lectin-like adhesins that bind to the intestinal wall. However, EHEC-related lectin-glycan interactions (LGIs) remain unknown. Here, we conducted a genome-wide investigation of putative adhesins to construct an LGI network. We performed microarray-based transcriptomic and proteomic analyses with E. coli EDL933. Using PSORTb-based analysis, potential outer-membrane-embedded adhesins were predicted from the annotated genes of 318 strains. Predicted proteins were classified using TMHMM v2.0, SignalP v5.0, and LipoP v1.0. Functional and protein-protein interaction analyses were performed using InterProScan and String databases, respectively. Structural information of lectin candidate proteins was predicted using Iterative Threading ASSEmbly Refinement (I-TASSER) and Spatial Epitope Prediction of Protein Antigens (SEPPA) tools based on 3D structure and B-cell epitopes. Pathway analysis returned 42,227 Gene Ontology terms; we then selected 2585 lectin candidate proteins by multi-omics analysis and performed homology modeling and B-cell epitope analysis. We predicted a total of 24,400 outer-membrane-embedded proteins from the genome of 318 strains and integrated multi-omics information into the genomic information of the proteins. Our integrated multi-omics data will provide a useful resource for the construction of LGI networks of E. coli.

Intestine specific regulation of pig cytidine-5'-monophospho-N-acetylneuraminic acid hydroxylase gene for N-glycolylneuraminic acid biosynthesis.

N-glycolylneuraminic acid (Neu5Gc), a generic form of sialic acid, is enzymatically synthesized by cytidine-5'-monophospho-N-acetylneuraminic acid hydroxylase (CMAH). Although expression of pig CMAH gene pcmah encoding CMAH has been reported to be regulated by pathogenic infection and developmental processes, little is known about the mechanisms underlying the regulation of pcmah gene expression. The objective of this study was to determine mechanism(s) involved in intestine specific regulation of pcmah gene by identifying several cis-acting elements and nuclear transcription factors that could directly interact with these cis-acting elements. We identified intestine specific promoter region (Pi) of pcmah gene located at upstream regions of the 5'flanking region of exon 1a and found that the promoter region is responsible for the transcriptional regulation of 5'pcmah-1. Based on reporter assays using serially constructed luciferase genes with each deleted promoter, we demonstrated that the Pi promoter activity was more active in intestinal IPI-2I cells than that in kidney PK15 cells, corresponding to both mRNA expression patterns in the two cell lines. In addition, we found that Sp1 transcription factor was necessary for basal activity of Pi promoter and that Ets-1 contributed to intestine-specific activity of Pi promoter. This study helps us understand transcriptional regulation of pcmah in the intestine of pig tissues. It also allows us to consider potential roles of Neu5Gc in interaction with environmental factors present in the intestinal tissue during pathogenic infection and developmental process.

Comparative genomic analysis of enterotoxigenic Escherichia coli O159 strains isolated from diarrheal patients in Korea.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a common cause of bacterial infection that leads to diarrhea. Although some studies have proposed a potential association between the toxic profile and genetic background, association between toxin of ETEC and phylo-group has not been reported yet. The objective of this study was to examine genomic and phylogenetic characteristics of ETEC strain NCCP15731 and NCCP15733 by whole genome sequencing and comparative genomic analysis of two phylo-groups of O159 reference strains. RESULTS: Whole genome sequencing showed that genome size of NCCP15731 strain was 4,663,459 bp, containing 4435 CDS and 19 RNAs. The genome size of NCCP15733 was 4,645,336 bp, containing 4369 CDS and 23 RNAs. Both NCCP15731 and NCCP15733 were classified in the phylo-group A, which is one of major E. coli phylogenetic groups. Their serotype was O159:H34. They possessed the virulence factor such as adherence systems, auto transporter systems, and flagella segments of major driving force for ETEC pathogenicity. They also harbored STh enterotoxin. Hierarchical clustering result based on the presence or absence of a total of 108 major virulence factors of 14 O159 ETEC strains showed that seven strains in phylo-group A and seven strains in phylo-group B1 were clustered each other, respectively. Colonization factors (CFs) of NCCP15731 or NCCP15733 were not detected. CONCLUSIONS: Serotype of NCCP15731 and NCCP15733, representing major types of ETEC in Korea, was O159:H34 and their MLST type was ST218. Comparison with other O159 strains revealed that NCCP15731 was specialized for transporter system and secretion system whereas NCCP15733 had unique genes related to capsular polysaccharide. Compared with E159, the most recent common ancestor, these two strains had different toxin type and virulence factors. These results will improve our understanding of ETEC O159 strains to prevent ETEC disease.

Esculentoside B inhibits inflammatory response through JNK and downstream NF-κB signaling pathway in LPS-triggered murine macrophage RAW 264.7 cells.

Natural compound esculentoside B (EsB), (2S,4aR,6aR,6aS,6bR,8aR,9R,10R,11S,12aR,14bS)-11-hydroxy-9-(hydroxymethyl)-2 methoxycarbonyl-2,6a,6b,9,12a-pentamethyl-10-[(2S,3R,4S,5R)-3,4,5-trihydroxyoxan-2-yl]oxy-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylic acid with molecular weight of 664.833, isolated from roots of Phytolacca acinosa Roxb has been widely used as a constituent of traditional Chinese medicine (TCM). However, the anti-inflammatory capacity of EsB has not been reported yet. Therefore, the objective of this study was to investigate anti-inflammatory activities of EsB in LPS-treated macrophage RAW 264.7 cells. EsB could inhibit nitric oxide (NO) production. EsB also suppressed gene and protein expression levels of inducible isoform of NO synthase (NOS) and cyclooxygenase-2 in a dose-dependent manner. In addition, EsB decreased gene expression and protein secretion levels of pro-inflammatory cytokines such as IL-1ß, TNF-α, and IL-6. EsB remarkably suppressed nuclear translocation of nuclear factor kappa-B (NF-κB) from cytosolic space. Phosphorylation of IκB was also inhibited by EsB. Moreover, EsB specifically down-regulated phospho-c-Jun N-terminal kinase (p-JNK), but not p-p38 or phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2). Taken together, these results suggest that EsB has inhibitory effect on inflammatory response by inactivating NF-κB and p-JNK. It could be used as a new modulatory drug for effective treatment of inflammation-related diseases.

Esculentoside H inhibits colon cancer cell migration and growth through suppression of MMP-9 gene expression via NF-kB signaling pathway.

A water-soluble saponin, Esculentoside H (EsH), 3-O-(O-ß-d-glucopyranosyl-(1→4)-ß-d-xylopyranosyl)-28-ß-d-glucopyranosylphytolaccagenin has been isolated and purified from the root extract of perennial plant Phytolacca esculenta. EsH is known to be an anticancer compound, having a capacity for TNF-α release. However, the effects of EsH on migration and growth in tumor cells have not yet been reported. In the current study, the suppressive effects of EsH on phorbol 12-myristate 13-acetate (PMA)-induced cell migration were examined in murine colon cancer CT26 cells and human colon cancer HCT116 cells. Interestingly, the transwell assay and wound healing show that EsH suppresses the PMA-induced migration and growth potential of HCT116 and CT26 colon cancer cells, respectively. EsH dose-dependently suppressed matrix metalloproteinases-9 (MMP-9) expression that was upregulated upon PMA treatment in messenger RNA levels and protein secretion. Since the expression of MMP-9 is correlated with nuclear factor-κB (NF-κB) signaling, it has been examined whether EsH inhibits PMA-induced IκB phosphorylation that leads to the suppression of NK-κB nuclear translocation. EsH repressed the phosphorylation level of JNK, but not extracellular signal-regulated kinase and p38 signaling when the cells were treated with PMA. Overall, these results demonstrated that EsH could suppress cancer migration through blockage of the JNK1/2 and NF-κB signaling-mediated MMP-9 expression.

Corrigendum to "Epidemiological Characterization of Imported Systemic Mycoses Occurred in Korea" [Osong Public Health Res Perspect 2018;9(5):255-60].

[This corrects the article on p. 255 in vol. 9, PMID: 30402381.].

Epidemiological Characterization of Imported Systemic Mycoses Occurred in Korea.

OBJECTIVES: Imported systemic mycoses is a severe fungal infection that can cause diseases in healthy people. However, there is a serious lack of epidemiological data about imported systemic mycoses. Therefore, an epidemiological characterization of imported systemic mycoses in Korea was performed. METHODS: We collected health insurance data between 2008 and 2012 from the Health Insurance Corporation and analyzed the data to determine the prevalence and treatment management of imported systemic mycoses. RESULTS: The prevalence of imported systemic mycoses between 2008 and 2012 increased slowly by 0.49/100,000 to 0.53/100,000 persons. The prevalence of coccidioidomycosis increased from 0.28/100,000 in 2008 to 0.36/100,000 persons in 2012. A mean of 229.6 cases occurred each year. Children and the elderly showed higher prevalence than adults in the 20- to 59-year-old age group. The rate of infection according to region ranged from 0.18/100,000 persons in Ulsan, to 0.59/100,000 persons in Gyeonggi. The prevalence in females was higher than that in males. Inpatient treatment was 3.3% (38 cases), with 96.7% treated as outpatients. Hospitalizations cost 272.7 million won and outpatient treatments cost 111.7 million won. The treatment cost for coccidioidomycosis from 2008 to 2012 was 330.9 million won, with personal charges of 79.2 million won and insurance charges of 251.7 million won. Most of the expenses for the coccidioidomycosis treatment were for inpatient treatment. CONCLUSION: The results in this study may be a useful resource for determining the changes in the trend of imported systemic mycoses.

Prevalence of Antibiotic Residues and Antibiotic Resistance in Isolates of Chicken Meat in Korea.

The aim of study was to investigate the correlation between the level of 17 antibiotic residues and 6 antibiotic resistances of Escherichia coli isolates in chicken meats. A total of 58 chicken meats were collected from retail grocery stores in five provinces in Korea. The total detection rate of antibiotic residues was 45% (26 out of 58). Ten out of 17 antibiotics were detected in chicken meats. None of the antibiotics exceeded the maximum residue level (MRLs) in chicken established by the Ministry of Food and Drug Safety (MFDS). The most detected antibiotics were amoxicillin (15.5%), followed by enrofloxacin (12.1%) and sulfamethoxazole (10.3%). In a total of 58 chicken meats, 51 E. coli strains were isolated. E. coli isolates showed the highest resistance to ampicillin (75%), followed by tetracycline (69%), ciprofloxacin (65%), trimethoprim/ sulfamethoxazole (41%), ceftiofur (22%), and amoxicillin/clavulanic acid (12%). The results of study showed basic information on relationship between antibiotic residue and resistance for 6 compounds in 13 chicken samples. Further investigation on the antibiotic resistance patterns of various bacteria species is needed to improve food safety.

Ganglioside GM3 suppresses lipopolysaccharide-induced inflammatory responses in rAW 264.7 macrophage cells through NF-κB, AP-1, and MAPKs signaling.

Gangliosides are known to specifically inhibit vascular leukocyte recruitment and consequent interaction with the injured endothelium, the basic inflammatory process. In this study, we have found that the production of nitric oxide (NO), a main regulator of inflammation, is suppressed by GM3 on murine macrophage RAW 264.7 cells, when induced by LPS. In addition, GM3 attenuated the increase in cyclooxyenase-2 (COX-2) protein and mRNA levels in lipopolysaccharide (LPS)-activated RAW 264.7 cells in a dose-dependent manner. Moreover, GM3 inhibited the expression and release of pro-inflammatory cytokines of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) in RAW 264.7 macrophages. At the intracellular level, GM3 inhibited LPS-induced nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein (AP)-1 in RAW 264.7 macrophages. We, therefore, investigated whether GM3 affects mitogen-activated protein kinase (MAPK) phosphorylation, a process known as the upstream signaling regulator. GM3 dramatically reduced the expression levels of the phosphorylated forms of ERK, JNK, and p38 in LPS-activated RAW 264.7 cells. These results indicate that GM3 is a promising suppressor of the vascular inflammatory responses and ganglioside GM3 suppresses the LPS-induced inflammatory response in RAW 264.7 macrophages by suppression of NF-κB, AP-1, and MAPKs signaling. Accordingly, GM3 is suggested as a beneficial agent for the treatment of diseases that are associated with inflammation.

Characterization of Clostridium difficile Strains Isolated from Patients with C. difficile-associated Disease in Korea.

OBJECTIVES: Studies on Clostridium difficile are rare in Korea. We investigated the epidemiological characteristics of C. difficile isolates from patients with C. difficile-associated disease (CDAD) in Korea. METHODS: Multiplex polymerase chain reaction was performed to detect the presence of tcdA and tcdB toxin genes. Antimicrobial susceptibility test was carried out by the disk-dilution method. C. difficile strains were subtyped by automated repetitive-element palindromic PCR (rep-PCR). RESULTS: Among patients with CDAD, 73 (25.8%), 32 (11.3%), 32 (11.3%), and 26 (9.2%) suffered from pneumonia, cancer or neoplasm, diabetes, and colitis, respectively. Of all stool samples, 43 samples (15.2%) were positive for C. difficile strains. We observed two expression patterns of toxin genes: tcdA+/tcdB+ (86% isolates) and tcdA-/tcdB+ (14% isolates), with all isolates expressing tcdB. Furthermore, some isolates were resistant to clindamycin (65%), ampicillin (56%), and cefazolin (40%), but all were susceptible to vancomycin and metronidazole. The tested samples were classified into diverse clusters using automated rep-PCR. CONCLUSION: Our findings revealed the characteristics and antibiotic resistance of C. difficile isolates from patients in Korea. The epidemiological data may provide valuable insight into development of treatment strategies for C. difficile infections in Korea.