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We previously reported potent ligands and inhibitors ofMycobacterium tuberculosisdethiobiotin synthetase (MtDTBS), a promising target for antituberculosis drug development (Schumannet al.,ACS Chem Biol. 2021, 16, 2339-2347); here the unconventional origin of the fragment compound they were derived from is described for the first time. Compound1(9b-hydroxy-6b,7,8,9,9a,9b-hexahydrocyclopenta[3,4]cyclobuta[1,2-c]chromen-6(6aH)-one), identified byin silicofragment screen, was subsequently shown by surface plasmon resonance to have dose-responsive binding (KD0.6 mM). Clear electron density was revealed in the DAPA substrate binding pocket, when1was soaked intoMtDTBS crystals, but the density was inconsistent with the structure of1. Here we show the lactone of1hydrolyses to carboxylic acid2under basic conditions, including those of the crystallography soak, with subsequent ring-opening of the component cyclobutane ring to form cyclopentylacetic acid3. Crystals soaked directly with authentic3produced electron density that matched that of crystals soaked with presumed1, confirming the identity of the bound ligand. The synthetic utility of fortuitously formed3enabled subsequent compound development into nanomolar inhibitors. Our findings represent an example of chemical modification within drug discovery assays and demonstrate the value of high-resolution structural data in the fragment hit validation process.SynopsisA molecule flagged in anin silicodocking screen againstMtDTBS, was inadvertently hydrolysed in the crystal conditions used for hit validation. The resulting fragment-sized molecule bound to the DAPA substrate binding pocket of the target enzyme (MtDTBS) with millimolar affinity, as measured by surface plasmon resonance, but was later modified to a highly potent (nanomolar) ligand and promising lead for the development of novel tuberculosis treatments.Graphical Abstract

The emergence of vancomycin-resistant Staphylococcus aureus (VRSA) threatens global health. The mechanism of vancomycin resistance of VRSA without vanA gene acquisition was not fully elucidated. Therefore, we aimed to determine the mechanism of vancomycin resistance of VRSA besides that by vanA gene acquisition. In this study, we obtained vancomycin-resistant strains (V036-V64; MIC = 64 µg /ml) from susceptible strain (V036; MIC = 0.5 µg /ml) by exposure of vancomycin in vitro and examined the phenotypic characteristics and antibiotic susceptibility profiles of the resistant strain (V036-V64). To identify the genetic variations caused vancomycin resistance, we determined the complete genome sequences of V036 and V036-V64 and analyzed for single-nucleotide polymorphisms (SNPs) between two strains. Morphologically, V036-V64 had a twofold thicker cell wall compared with V036. Linezolid, rifampicin, and ceftaroline had similar MIC ranges against V036-V64 and V036, but V036-V64 showed lower susceptibilities to daptomycin and telavancin. We detected eight single-nucleotide polymorphisms differing between V036-V64 and V036: rimM (G16D), ssaA2 (G128A), rpsK (P60R), rpoB (R917C), walK (T492R), d-alanyl-d-alanine carboxypeptidase (L307I), vraT (A152V), and chromosome segregation ATPase (T440I). This study demonstrates that, under selective pressure, by the accumulation of mutations in genes related to cell wall synthesis, vancomycin-susceptible S. aureus can develop thicker cell walls and, hence, develop high vancomycin resistance. Thus, we highlight a novel vanA-negative mechanism for VRSA emergence.

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-β-lactamase, oxacillinase, imipenem-hydrolyzing, Verona integron-encoded metallo-β-lactamase, and Guiana extended-spectrum β-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.

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.

The emergence of third-generation cephalosporin resistance in Escherichia coli is increasing at an alarming rate in many countries. Thus, the aim of this study was to analyze co-infecting bla CTX-M-producing pathogenic E. coli isolates linked to three school outbreaks. Among 66 E. coli isolates, 44 were identified as ETEC O25, an ETEC isolate serotype was O2, and the other 21 were confirmed as EAEC O44. Interestingly, six patients were co-infected with EAEC O44 and ETEC O25. For these isolates, molecular analysis [antibiotic susceptibility testing, identification of the β-lactamase gene, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE)] was performed for further characterization. In addition, the transmission capacity of bla CTX-M genes was examined by conjugation experiments. Whole-genome sequencing (WGS) was performed on representative EAEC O44 and ETEC O25 isolates associated with co-infection and single-infection. All isolates were resistant to cefotaxime and ceftriaxone. All EAEC isolates carried the bla CTX-M-14 gene and all ETEC isolates the bla CTX-M-15 gene, as detected by multiplex PCR and sequencing analysis. Sequence type and PFGE results indicated three different patterns depending on the O serotype. WGS results of representative isolates revealed that the ETEC O25 strains harbored bla CTX-M-15 located on IncK plasmids associated with the Δbla TEM-bla CTX-M-15-orf477 transposon. The representative EAEC O44 isolates carried bla CTX-M-14 on the chromosome, which was surrounded by the ISEcp1-bla CTX-M-14-IS903 transposon. To the best of our knowledge, this is the first report of co-infection with chromosomally located bla CTX-M-14 and plasmid-encoding bla CTX-M-15 in pathogenic E. coli. Our findings indicate that resistance genes in clinical isolates can spread through concurrent combinations of chromosomes and plasmids.

Background The emerging mobile colistin resistance gene, mcr-1, is an ongoing worldwide concern and an evaluation of clinical isolates harboring this gene is required in Korea. We investigated mcr-1-possessing Enterobacteriaceae among Enterobacteriaceae strains isolated in Korea, and compared the genetic details of the plasmids with those in Escherichia coli isolates from livestock. Methods Among 9,396 Enterobacteriaceae clinical isolates collected between 2010 and 2015, 1,347 (14.3%) strains were resistant to colistin and those were screened for mcr-1 by PCR. Colistin minimum inhibitory concentrations (MICs) were determined by microdilution, and conjugal transfer of the mcr-1-harboring plasmids was assessed by direct mating. Whole genomes of three mcr-1-positive Enterobacteriaceae clinical isolates and 11 livestock-origin mcr-1-positive E. coli isolates were sequenced. Results Two E. coli and one Enterobacter aerogenes clinical isolates carried carried IncI2 plasmids harboring mcr-1, which conferred colistin resistance (E. coli MIC, 4 mg/L; E. aerogenes MIC, 32 mg/L). The strains possessed the complete conjugal machinery except for E. aerogenes harboring a truncated prepilin peptidase. The E. coli plasmid transferred more efficiently to E. coli than to Klebsiella pneumoniae or Enterobacter cloacae recipients. Among the three bacterial hosts, the colistin MIC was the highest for E. coli owing to the higher mcr-1-plasmid copy number and mcr-1 expression levels. Ten mcr-1-positive chicken-origin E. coli strains also possessed mcr-1-harboring IncI2 plasmids closely related to that in the clinical E. aerogenes isolate, and the remaining one porcine-origin E. coli possessed an mcr-1-harboring IncX4 plasmid. Conclusions mcr-1-harboring IncI2 plasmids were identified in clinical Enterobacteriaceae isolates. These plasmids were closely associated with those in chicken-origin E. coli strains in Korea, supporting the concept of mcr-1 dissemination between humans and livestock.

Aim: To assess the epidemiology of carbapenemase-producing Enterobacteriaceae (CPE) in South Korea. Materials & methods: From 2011 to 2015, 2487 carbapenem-nonsusceptible Enterobacteriaceae were collected through the Korean National Laboratory Surveillance System. Disk-diffusion for antimicrobial susceptibility, PCR/sequencing to detect carbapenemase genes and multilocus sequence typing for molecular epidemiology were carried out. Results: The number of carbapenem-nonsusceptible Enterobacteriaceae was increasing approximately 1.5-fold per year and the proportion of CPEs was exponentially confirmed from 2014. KPC was the most dominant, mostly associated with Klebsiella pneumoniae ST11 and ST307, NDM was the second and OXA-48-like was the third dominant carbapenemases. The IMP, VIM and GES-5 CPEs were identified sporadically. Conclusion: The nation-wide spreads of KPC, NDM and OXA-48-like CPEs were in an alarming epidemiological stage.

Background Methicillin-resistant Staphylococcus aureus (MRSA) are important pathogens causing nosocomial infections in Korean hospitals. This study aimed to investigate the epidemiological and genetic diversity of clinical S. aureus isolates in healthcare settings from 2001 to 2008. Methods Samples and data were obtained from 986 individuals as part of the National Antimicrobial Surveillance Project, involving 10 regions nationwide. Molecular typing studies, including multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing were performed, and a representative clone of Korean MRSA was classified by combinational grouping using a DiversiLab (DL; bioMérieux, France) repetitive element polymerase chain reaction (rep-PCR) system. Results Nine Korean MRSA clones (KMRSA-1 to -9) were identified by analysis of genetic backgrounds and molecular characteristics. KMRSA-1 to -3, expressing clonal complex (CC) 5 (carrying SCCmec II), CC8 (carrying SCCmec III), and CC72 (carrying SCCmec IV) were spread nationwide. In contrast, KMRSA-6 was highly prevalent in Gyeongsangnam-do, and KMRSA-4 was highly prevalent in Jeollanam-do and Jeollabuk-do. Conclusions Epidemic KMRSA clones were genetically similar to major clones identified from the USA, with the exception of KMRSA-2, which had the SCCmec III type. Our results provide important insights into the distribution and molecular genetics of MRSA strains in Korea and may aid in the monitoring of MRSA spread throughout the country.

Lee, K.-J. and Cho, Y.-S., 2020. Focusing of tsunami energy at Imwon Port. In: Malvarez, G. and Navas, F. (eds.), Global Coastal Issues of 2020. Journal of Coastal Research, Special Issue No. 95, pp. 247–251. Coconut Creek (Florida), ISSN 0749-0208.In the last few decades, several catastrophic tsunamis triggered by undersea earthquakes have occurred and struck not only neighboring but also distant coastal communities around the world. These tsunamis have caused a large number of human casualties and severe property damage. In this study, a geographical feature of the Imwon Port located at eastern coast of the Korean Peninsula, is investigated by studying a group of historical and virtual tsunamis caused by 3 historical and 11 virtual undersea earthquakes suggested by KEDO (1999). In addition, the effectiveness of the wave ray model is then examined by estimating tsunami heights at specific positions. Finally, the location of wave energy focusing is predicted by estimating tsunami heights and wave ray tracing, and whether the location is right is reconfirmed by the actual particle velocity vector map. The results of this study shall be used by a civil defense authority to mitigate the tsunami hazards in the case of a real tsunami attack.

Objectives To investigate the risk factors of patients with Klebsiella pneumoniae (KP) bloodstream infection (BSI) with a focus on antimicrobial resistance and virulence factors. Methods All KP BSI patients (n = 579) from six general hospitals during a 1 year period were included in this study. The risk factors of hosts and causative KP isolates were assessed to determine associations with the 30 day mortality of KP BSI patients by multivariate Cox hazards modelling. Results The 30 day mortality rate of KP BSI patients was 16.9% (98/579). Among the host-associated factors, increased SOFA score and leucopenia status exhibited strong associations with increased 30 day mortality. Among the pathogenic factors, carriage of the pks gene cluster (adjusted HR 1.80; 95% CI 1.16-2.79) was a risk factor, especially when accompanied by MDR. In this regard, KP isolates of the wzi50 capsular type (n = 22) frequently harboured pks (63.6%, 14/22) and ybtA (68.2%, n = 15) and mostly exhibited MDR (63.6%, n = 14), resulting in increased 30 day mortality. In contrast, hypermucoviscous KP isolates showed an inverse association with 30 day mortality (adjusted HR 0.55; 95% CI 0.33-0.90). Conclusions Despite the reported virulence of hypermucoviscous KP strains, they were associated with good prognoses in KP BSI patients. Importantly, carriage of the pks gene cluster, which is responsible for the synthesis of colibactin, was a relevant marker of early mortality.

A total of 281 nonduplicated Staphylococcus aureus blood isolates were collected from January to May 2017 from eight hospitals in South Korea to investigate the epidemiological traits of ceftaroline resistance in methicillin-resistant S. aureus (MRSA). Cefoxitin-disk diffusion tests and the mecA gene PCR revealed that 56.6% (159/281) of the S. aureus isolates were MRSA, and most belonged to ST5 (50.3%, 80/281) and ST72 (41.5%, 66/281). Of the MRSA isolates, 44.0% (70/159) were nonsusceptible to ceftaroline (MIC ≥ 2 mg/liter), whereas all of the methicillin-susceptible S. aureus isolates were susceptible to the drug. Eight amino acid substitutions in penicillin-binding protein 2a (PBP2a), including four (L357I, E447K, I563T, and S649A) in the penicillin-binding domain (PBD) and four (N104K, V117I, N146K, and A228V) in the non-PBD (nPBD) of PBP2a, were associated with ceftaroline resistance. The accumulation of substitutions in PBP2a resulted in the elevation of ceftaroline MICs: one substitution at 1 to 2 mg/liter, two or three substitutions at 2 to 4 mg/liter, and five substitutions at 4 or 16 mg/liter. Ceftaroline resistance in MRSA might be the result of clone-specific PBP2a polymorphism, along with substitutions both in PBD and nPBD, and the elevated ceftaroline MICs were associated with the substitution sites and accumulation of substitutions.

This study was carried out to investigate the epidemiological time-course of New Delhi metallo-beta-lactamase- (NDM-) mediated carbapenem resistance in Enterobacteriaceae in South Korea. A total of 146 non-duplicate NDM-producing Enterobacteriaceae recovered between 2010 and 2015 were voluntarily collected from 33 general hospitals and confirmed by PCR. The species were identified by sequences of the 16S rDNA. Antimicrobial susceptibility was determined either by the disk diffusion method or by broth microdilution, and the carbapenem MICs were determined by agar dilution. Then, multilocus sequence typing and PCR-based replicon typing was carried out. Co-carried genes for drug resistance were identified by PCR and sequencing. The entire genomes of eight random selected NDM producers were sequenced. A total of 69 Klebsiella pneumoniae of 12 sequence types (STs), 34 Escherichia coli of 15 STs, 28 Enterobacter spp. (including one Enterobacter aerogenes), nine Citrobacter freundii, four Raoultella spp., and two Klebsiella oxytoca isolates produced either NDM-1 (n = 126), NDM-5 (n = 18), or NDM-7 (n = 2). The isolates co-produced CTX-M-type ESBL (52.1%), AmpCs (27.4%), additional carbapenemases (7.1%), and/or 16S rRNA methyltransferases (4.8%), resulting in multidrug-resistance (47.9%) or extensively drug-resistance (52.1%). Among plasmids harboring blaNDM, IncX3 was predominant (77.4%), followed by the IncFII type (5.8%). Genome analysis revealed inter-species and inter-strain horizontal gene transfer of the plasmid. Both clonal dissemination and plasmid transfer contributed to the wide dissemination of NDM producers in South Korea.

The Korean government established an antimicrobial resistance (AMR) surveillance system, compatible with the Global AMR Surveillance System (GLASS): Kor-GLASS. We describe results from the first year of operation of the Kor-GLASS from May 2016 to April 2017, comprising all non-duplicated clinical isolates of major pathogens from blood, urine, faeces and urethral and cervical swabs from six sentinel hospitals. Antimicrobial susceptibility tests were carried out by disk diffusion, Etest, broth microdilution and agar dilution methods. Among 67,803 blood cultures, 3,523 target pathogens were recovered. The predominant bacterial species were Escherichia coli (n = 1,536), Klebsiella pneumoniae (n = 597) and Staphylococcus aureus (n = 584). From 57,477 urine cultures, 6,394 E. coli and 1,097 K. pneumoniae were recovered. Bloodstream infections in inpatients per 10,000 patient-days (10TPD) were highest for cefotaxime-resistant E. coli with 2.1, followed by 1.6 for meticillin-resistant Sta. aureus, 1.1 for imipenem-resistant Acinetobacter baumannii, 0.8 for cefotaxime-resistant K. pneumoniae and 0.4 for vancomycin-resistant Enterococcus faecium. Urinary tract infections in inpatients were 7.7 and 2.1 per 10TPD for cefotaxime-resistant E. coli and K. pneumoniae, respectively. Kor-GLASS generated well-curated surveillance data devoid of collection bias or isolate duplication. A bacterial bank and a database for the collections are under development.

Surveillance plays a pivotal role in overcoming antimicrobial resistance (AMR) in bacterial pathogens, and a variety of surveillance systems have been set up and employed in many countries. In 2015, the World Health Organization launched the Global Antimicrobial Resistance Surveillance System (GLASS) as a part of the global action plan to enhance national and global surveillance and research. The aims of GLASS are to foster development of national surveillance systems and to enable collection, analysis and sharing of standardised, comparable and validated data on AMR between different countries. The South Korean AMR surveillance system, Kor-GLASS, is compatible with the GLASS platform and was established in 2016 and based on the principles of representativeness, specialisation, harmonisation and localisation. In this report, we summarise principles and processes in order to share our experiences with other countries planning to establish a national AMR surveillance system. The pilot operation of Kor-GLASS allowed us to understand the national burden of specific infectious diseases and the status of bacterial AMR. Issues pertaining to high costs and labour-intensive operation were raised during the pilot, and improvements are being made.

Between 2014 and 2015, the Klebsiella pneumoniae carbapenemase (KPC) was becoming endemic in South Korea. To assess this period of transition, we analyzed KPC producers in terms of molecular epidemiology. A total of 362 KPC-producing Enterobacteriaceae strains, including one from 2013, 13 from 2014, and 348 from 2015, were actively collected from 60 hospitals throughout the peninsula. Subtypes of KPC were determined by PCR and direct sequencing, and isotypes of Tn4401 (the transposon flanking the blaKPC gene) were specified by PCR using isotype-specific primers and direct sequencing. Sporadic occurrence of KPC-producing Enterobacteriaceae was initially observed around Seoul, which is the most crowded district of the country, and these strains rapidly disseminated in 2014, to the other parts of the country in 2015. The bacterial clones responsible for the extreme epidemiological transition were K. pneumoniae ST307 (46.2%) and ST11 (21.3%). Less frequently, E. coli (4.7%), Enterobacter spp. (1.4%), and other Enterobacteriaceae members (1.7%) producing the enzyme were identified. The blaKPC-2 gene bracketed by Tn4401a (72.1%) was the most prevalent mobile genetic element responsible for the dissemination, and the same gene carried either by Tn4401b (2.2%) or Tn4401c (6.6%) was identified at a lesser frequency. The genes blaKPC-3 (1.6%) and blaKPC-4 (6.4%), both flanked by Tn4401b, were occasionally identified. This study showed endemic dissemination of KPC producers in 2015 due to a clonal spread of two K. pneumoniae strains. Further systemic surveillance is needed to monitor dissemination of KPC-producers.

Objectives High rates of carbapenem resistance in the human pathogen Acinetobacter baumannii threaten public health and need to be scrutinized. Methods A total of 356 A. baumannii and 50 non-baumannii Acinetobacter spp. (NBA) strains collected in 2013 throughout South Korea were studied. The type of blaOXA-23 transposon was determined by PCR mapping and molecular epidemiology was assessed by MLST. Twelve representative strains and two comparative A. baumannii were entirely sequenced by single-molecule real-time sequencing. Results The carbapenem resistance rate was 88% in A. baumannii, mainly due to blaOXA-23, with five exceptional cases associated with ISAba1-blaOXA-51-like. The blaOXA-23 gene in A. baumannii was carried either by Tn2006 (44%) or Tn2009 (54%), with a few exceptions carried by Tn2008 (1.6%). Of the NBA strains, 14% were resistant to carbapenems, two with blaOXA-58 and five with blaOXA-23 associated with Tn2006. The Tn2006-possessing strains belonged to various STs, whereas Tn2008- and Tn2009-possessing strains were limited to ST208 and ST191, respectively. The three transposons were often multiplied in the chromosome, and the gene copy number and the carbapenem MICs presented linear relationships either very strongly for Tn2008 or moderately for Tn2006 and Tn2009. Conclusions The dissemination of Tn2006 was facilitated by its capability for intercellular transfer and that of Tn2009 was attributable to successful dissemination of the ST191 bacterial host carrying the transposon. Tn2008 was infrequent because of its insufficient ability to undergo intercellular transfer and the scarce bacterial host A. baumannii ST208. Gene amplification is an adaptive mechanism for bacteria that encounter antimicrobial drugs.

We investigated changes in serotypes and antimicrobial susceptibilities among 386 isolates of invasive Streptococcus pneumoniae collected from numerous hospitals in Korea from 1996 to 2008. Serotypes 19F (9.8 %), 23F (8.3 %), 19A (7.8 %), 6A (7.5 %), 3 (7.3 %), 9V (6.5 %), 6B (6.2 %), 14 (4.9 %), 1 (3.9 %), 11A (3.9 %) and 4 (3.1 %) represented 69.2 % of all isolates. While the overall proportion of PCV7 serotypes was stable over time, we observed modest decreases in children S. pneumoniae isolates showed high resistance rates to erythromycin (74.9 %), tetracycline (71.1 %) and clindamycin (61.7 %). Between 1996–2003 and 2004–2008, non-susceptibility rates to cefotaxime and multi-drugs (three or more classes) in PCV7 serotypes showed a declining trend, while in non-PCV7 serotypes there was an increasing trend. Non-PCV7 serotypes 6A and 19A, which mostly exhibited multidrug-resistant phenotypes (69.0 % and 76.7 % respectively), increased between 1996–2003 and 2004–2008. Although PCV7 was introduced in Korea in November 2003, pneumococcal vaccination has not been included in the national child vaccination programme. Our results provide details of serotype occurrence that will be useful when adoption of universal pneumococcal vaccination in Korea is being considered.

Aminoglycosides are key drugs for the treatment of multidrug-resistant tuberculosis. A total of 97 extensively drug-resistant (XDR) and 29 pan-susceptible Mycobacterium tuberculosis isolates from Korean tuberculosis patients were analyzed to characterize mutations within the rrs, rpsL, gidB, eis and tlyA genes. Thirty (56.6 %) of the 53 streptomycin (STR)-resistant strains had a rpsL mutation and eight strains (15.1 %) had a rrs (514 or 908 site) mutation, whereas 11 (20.8 %) of the 53 STR-resistant strains had a gidB mutation without rpsL or either rrs mutation. Most of the gidB mutations conferred low-level STR resistance, and 22 of these mutations were novel. Mutation at position 1401 in rrs lead to resistance to kanamycin (80/95 = 84.2 %; KAN), amikacin (80/87 = 92.0 %; AMK), and capreomycin (74/86 = 86.0 %; CAP). In this study, 13.7 % (13/95) of KAN-resistant strains showed eis mutations, including 4 kinds of novel mutations. Isolates with eis structural gene mutations were cross-resistant to STR, KAN, CAP, and AMK. Here, 5.8 % (5/86) of the CAP-resistant strains harbored a tlyA mutation that included 3 different novel point mutations. Detection of the A1401G mutation appeared to be 100 % specific for the detection of resistance to KAN and AMK. These data establish the presence of phenotypic XDR strains using molecular profiling and are helpful to understanding of aminoglycoside resistance at the molecular level.

In total, 301 isolates of Streptococcus pneumoniae collected from patients with respiratory tract infections admitted at primary clinics during 2002-2005 were tested for multidrug-resistance (MDR) phenotypes and their serotypes in Korea. The predominant serotypes were 19F, 19A, 23F, 11A, 3, 6A, and 6B, accounting for 67.8% of all isolates. Their serotype coverage by 23-valent polysaccharide vaccine and 7-valent conjugation vaccine was 73.1% and 39.2%, respectively. For the application of Clinical and Laboratory Standards Institute's new breakpoint for penicillin, the resistance rate of penicillin was 27.9% (but the penicillin resistance was 80.4% based on the previous breakpoint for penicillin of Clinical and Laboratory Standards Institute). Actually, the full resistance rate was only 4.0% (minimum inhibitory concentrationor=8 mg/L). Resistances to erythromycin, clindamycin, and tetracycline were very high (82.9%, 79.4%, and 71.7%, respectively). Especially, 56.1% of all the isolates were MDR, defined as resistant to three or more of the following agents: penicillin, erythromycin, clindamycin, cefotaxime, tetracycline, and levofloxacin. MDR strains were relatively associated with serotypes 19F, 19A, 23F, and 11A, accounting for 58.0% of the isolates. Their serotype coverage by 23-valent polysaccharide vaccine and 7-valent conjugation vaccine was 79.5% and 45.9%, respectively. Levofloxacin, as a representative fluoroquinolone, was active against 88.2% of all MDR isolates. Of particular concern was the high prevalence of MDR pneumococci in non-PCV7 serotypes with an MDR serotype 19A, 11A, 3, and 6A being mostly responsible. It would be prudent to consider more efficient protective strategies for people at high risk for pneumococcal diseases in regions with a high prevalence of MDR pneumococci.

Clonorchis sinensis, the Chinese liver fluke, is the causative agent of clonorchiasis as well as liver and biliary diseases. The excretory-secretory products (ESPs) of the parasites play important roles in host-parasite interactions. In this study, we have investigated the proteome of ESPs obtained from C. sinensis adult worms. Although the full genome database of C. sinensis is not yet available, we have successfully identified 62 protein spots using 2-DE-based mass analysis and EST database of C. sinensis. The proteins identified include detoxification enzymes, such as glutathione S-transferase and thioredoxin peroxidase, myoglobin and a number of cysteine proteases that are expressed abundantly. In order to identify potential targets for the diagnosis and therapy of clonorchiasis, we conducted immunoblot analysis of the ESPs proteome using the sera obtained from clonorchiasis patients and identified legumains and cysteine proteases as antigens present in the ESPs. Although the cysteine proteases were previously reported to elicit antigenicity, the legumains are found herein for the first time as a serological antigen of C. sinensis. To confirm these findings, we expressed recombinant legumain in Escherichia coli and verified that recombinant legumain also functions as a potent antigen against the sera of clonorchiasis patients. Our results illustrate the validity of immuno-proteomic approaches in the identification of serodiagnostic antigens in the parasites.

A prototypical case of a molecular radical cation is reported whose electrostatic charge distribution is determined entirely and uniquely by its conformational structures. Experimental observation of charge distribution in a molecular ion was for the first time demonstrated to be feasible by optical spectroscopy in the case of L-phenylalanine cation by utilizing the fact that its photodissociation propensity is entirely determined by the electronic character of its charge distribution. The cationic charge was explicitly shown to be localized in a single site or two different sites depending on the molecular conformation.

Botulinum neurotoxin type A (BoNT/A) is the most potent protein toxin and causes fatal flaccid muscle paralysis by blocking neurotransmission. Application of BoNT/A has been extended to the fields of therapeutics and biodefense. Nevertheless, the global response of host immune cells to authentic BoNT/A has not been reported. Employing microarray analysis, we performed global transcriptional profiling of RAW264.7 cells, a murine alveolar macrophage cell line. We identified 70 genes that were modulated following 1 nM BoNT/A treatment. The altered genes were mainly involved in signal transduction, immunity and defense, protein metabolism and modification, neuronal activities, intracellular protein trafficking, and muscle contraction. Microarray data were validated with real-time RT-PCR for seven selected genes including tlr2, tnf, inos, ccl4, slpi, stx11, and irg1. Proinflammatory mediators such as nitric oxide (NO) and tumor necrosis factor alpha (TNFα) were induced in a dose-dependent manner in BoNT/A-stimulated RAW264.7 cells. Increased expression of these factors was inhibited by monoclonal anti-Toll-like receptor 2 (TLR2) and inhibitors specific to intracellular proteins such as c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK). BoNT/A also suppressed lipopolysaccharide-induced NO and TNFα production from RAW264.7 macrophages at the transcription level by blocking activation of JNK, ERK, and p38 MAPK. As confirmed by TLR2-/- knock out experiments, these results suggest that BoNT/A induces global gene expression changes in host immune cells and that host responses to BoNT/A proceed through a TLR2-dependent pathway, which is modulated by JNK, ERK, and p38 MAPK.

Quality control (QC) processes are being performed in the majority of clinical microbiology laboratories to ensure the performance of microbial identification and antimicrobial susceptibility testing by using ATCC strains. To obtain these ATCC strains, some inconveniences are encountered concerning the purchase cost of the strains and the shipping time required. This study was focused on constructing a database of reference strains for QC processes using domestic bacterial strains, concentrating primarily on antimicrobial susceptibility testing. Three strains (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) that showed legible results in preliminary testing were selected. The minimal inhibitory concentrations (MICs) and zone diameters (ZDs) of eight antimicrobials for each strain were determined according to the CLSI M23. All resulting MIC and ZD ranges included at least 95% of the data. The ZD QC ranges obtained by using the CLSI method were less than 12 mm, and the MIC QC ranges extended no more than five dilutions. This study is a preliminary attempt to construct a bank of Korean QC strains. With further studies, a positive outcome toward cost and time reduction can be anticipated.

To improve the erythritol productivity ofPenicillium sp. KJ81, mutants were obtained using UV irradiation and NTG treatment. Among these mutants,Penicillium sp. KJ-UV29 revealed no morphological changes, yet was superior to the wild strain in the following three points: (1)Penicillium sp. KJ-UV29 produced more erythritol than the wild strain under the same conditions, (2) no foam was produced during cultivation, unlike the wild strain, and (3) the mutant produced a significantly lower amount of glycerol.Penicillium sp KJ-UV29 produced as much as 15.1 g/L of erythritol, whereas the wild-typePenicillium sp. KJ-UV29 produced as much as 15.1 g/L of erythritol, whereas the wild-typePenicillium sp. KJ81 only produced 11.7 g/L.Penicillium sp. KJ-UV29 only generated 6.1 g/L of glycerol, compared to 19.4 g/L produced by the wild strain. When investigating the optimal culture conditions for erythritol production by the mutant strainPenicillium sp. KJ-UV29, sucrose was idetified as the most effective carbon source, and the mutant was even able to produce erythritol in a 70% sucrose-containing medium, although a 30% sucrose medium exhibited the highest productivity. The production of erythritol byPenicillium sp. KJ-UV29 was also significantly increased by the addition of ammonium carbonate, potassium nitrate, and sodium nitrate. Accordingly, under optimal conditions,Penicillium sp. KJ-UV29 produced 45.2 g/L of erythritol in a medium containing 30% sucrose, 0.5% yeast extract, 0.5% (NH4)2C2O4 0.1% NaNO3, and 0.01% FeSO4 with 1 vvm aeration and 200 rpm agitation at 37°C for 7 days in a 5-L jar fermentor.