PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period.

PERIOD (PER) and Casein Kinase 1δ regulate circadian rhythms through a phosphoswitch that controls PER stability and repressive activity in the molecular clock. CK1δ phosphorylation of the familial advanced sleep phase (FASP) serine cluster embedded within the Casein Kinase 1 binding domain (CK1BD) of mammalian PER1/2 inhibits its activity on phosphodegrons to stabilize PER and extend circadian period. Here, we show that the phosphorylated FASP region (pFASP) of PER2 directly interacts with and inhibits CK1δ. Co-crystal structures in conjunction with molecular dynamics simulations reveal how pFASP phosphoserines dock into conserved anion binding sites near the active site of CK1δ. Limiting phosphorylation of the FASP serine cluster reduces product inhibition, decreasing PER2 stability and shortening circadian period in human cells. We found that Drosophila PER also regulates CK1δ via feedback inhibition through the phosphorylated PER-Short domain, revealing a conserved mechanism by which PER phosphorylation near the CK1BD regulates CK1 kinase activity.

Endogenous circadian reporters reveal functional differences of PERIOD paralogs and the significance of PERIOD:CK1 stable interaction.

Adverse consequences from having a faulty circadian clock include compromised sleep quality and poor performance in the short-term, and metabolic diseases and cancer in the long-term. However, our understanding of circadian disorders is limited by the incompleteness of our molecular models and our dearth of defined mutant models. Because it would be prohibitively expensive to develop live animal models to study the full range of complicated clock mechanisms, we developed PER1-luc and PER2-luc endogenous circadian reporters in a validated clock cell model, U-2 OS, where the genome can be easily manipulated, and functional consequences of mutations can be accurately studied. When major clock genes were knocked out in these cells, circadian rhythms were modulated similarly compared with corresponding mutant mice, validating the platform for genetics studies. Using these reporter cells, we uncovered critical differences between two paralogs of PER. Although PER1 and PER2 are considered redundant and either one can serve as a pacemaker alone, they were dramatically different in biochemical parameters such as stability and phosphorylation kinetics. Consistently, circadian phase was dramatically different between PER1 and PER2 knockout reporter cells. We further showed that the stable binding of casein kinase1δ/ε to PER is not required for PER phosphorylation itself, but is critical for delayed timing of phosphorylation. Our system can be used as an efficient platform to study circadian disorders associated with pathogenic mutations and their underlying molecular mechanisms.

Wake-sleep cycles are severely disrupted by diseases affecting cytoplasmic homeostasis.

The circadian clock is based on a transcriptional feedback loop with an essential time delay before feedback inhibition. Previous work has shown that PERIOD (PER) proteins generate circadian time cues through rhythmic nuclear accumulation of the inhibitor complex and subsequent interaction with the activator complex in the feedback loop. Although this temporal manifestation of the feedback inhibition is the direct consequence of PER's cytoplasmic trafficking before nuclear entry, how this spatial regulation of the pacemaker affects circadian timing has been largely unexplored. Here we show that circadian rhythms, including wake-sleep cycles, are lengthened and severely unstable if the cytoplasmic trafficking of PER is disrupted by any disease condition that leads to increased congestion in the cytoplasm. Furthermore, we found that the time delay and robustness in the circadian clock are seamlessly generated by delayed and collective phosphorylation of PER molecules, followed by synchronous nuclear entry. These results provide clear mechanistic insight into why circadian and sleep disorders arise in such clinical conditions as metabolic and neurodegenerative diseases and aging, in which the cytoplasm is congested.

Ceftaroline Resistance by Clone-Specific Polymorphism in Penicillin-Binding Protein 2a of Methicillin-Resistant Staphylococcus aureus.

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.

Readjustment of circadian clocks by exercise intervention is a potential therapeutic target for sleep disorders: a narrative review.

PURPOSE: Circadian clocks are evolved endogenous biological systems that communicate with environmental cues to optimize physiological processes, such as the sleep-wake cycle, which is nearly related to quality of life. Sleep disorders can be treated using pharmacological strategies targeting melatonin, orexin, or core clock genes. Exercise has been widely explored as a behavioral treatment because it challenges homeostasis in the human body and affects the regulation of core clock genes. Exercise intervention at the appropriate time of the day can induce a phase shift in internal clocks. Although exercise is a strong external time cue for resetting the circadian clock, exercise therapy for sleep disorders remains poorly understood. METHODS: This review focused on exercise as a potential treatment for sleep disorders by tuning the internal circadian clock. We used scientific paper depositories, including Google Scholar, PubMed, and the Cochrane Library, to identify previous studies that investigated the effects of exercise on circadian clocks and sleep disorders. RESULTS: The exercise-induced adjustment of the circadian clock phase depended on exercise timing and individual chronotypes. Adjustment of circadian clocks through scheduled morning exercises can be appropriately prescribed for individuals with delayed sleep phase disorders. Individuals with advanced sleep phase disorders can synchronize their internal clocks with their living environment by performing evening exercises. Exercise-induced physiological responses are affected by age, sex, and current fitness conditions. CONCLUSION: Personalized approaches are necessary when implementing exercise interventions for sleep disorders.

Predictive coding with spiking neurons and feedforward gist signaling.

Predictive coding (PC) is an influential theory in neuroscience, which suggests the existence of a cortical architecture that is constantly generating and updating predictive representations of sensory inputs. Owing to its hierarchical and generative nature, PC has inspired many computational models of perception in the literature. However, the biological plausibility of existing models has not been sufficiently explored due to their use of artificial neurons that approximate neural activity with firing rates in the continuous time domain and propagate signals synchronously. Therefore, we developed a spiking neural network for predictive coding (SNN-PC), in which neurons communicate using event-driven and asynchronous spikes. Adopting the hierarchical structure and Hebbian learning algorithms from previous PC neural network models, SNN-PC introduces two novel features: (1) a fast feedforward sweep from the input to higher areas, which generates a spatially reduced and abstract representation of input (i.e., a neural code for the gist of a scene) and provides a neurobiological alternative to an arbitrary choice of priors; and (2) a separation of positive and negative error-computing neurons, which counters the biological implausibility of a bi-directional error neuron with a very high baseline firing rate. After training with the MNIST handwritten digit dataset, SNN-PC developed hierarchical internal representations and was able to reconstruct samples it had not seen during training. SNN-PC suggests biologically plausible mechanisms by which the brain may perform perceptual inference and learning in an unsupervised manner. In addition, it may be used in neuromorphic applications that can utilize its energy-efficient, event-driven, local learning, and parallel information processing nature.

Molecular detection of Borrelia theileri in cattle in Korea.

Bovine borreliosis, caused by Borrelia theileri which is transmitted via hard tick bites, is associated with mild clinical symptoms, such as fever, lethargy, hemoglobinuria, anorexia, and anemia. Borrelia theileri infects various animals, such as cattle, deer, horses, goats, sheep, and wild ruminants, in Africa, Australia, and South America. Notably, no case of B. theileri infection has been reported in Korean cattle to date. In this study, 101 blood samples were collected from a Korean indigenous cattle breed, among which 1.98% tested positive for B. theileri via nested PCR. The obtained sequences exhibited high homology with B. theileri strains identified in other regions. Phylogenetic analysis of 16S rRNA confirmed the B. theileri group affiliation; however, flagellin B sequences exhibited divergence, potentially due to regional evolutionary differences. This study provides the first molecular confirmation of B. theileri infection in Korean livestock. Further isolation and nucleotide sequence analyses are necessary to better understand the presence of B. theileri strains in cows in Korea.

Molecular genetics of Mycobacterium tuberculosis resistant to aminoglycosides and cyclic peptide capreomycin antibiotics in Korea.

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.

Generation of CRISPR-Cas9-mediated knockin mutant models in mice and MEFs for studies of polymorphism in clock genes.

The creation of mutant mice has been invaluable for advancing biomedical science, but is too time- and resource-intensive for investigating the full range of mutations and polymorphisms. Cell culture models are therefore an invaluable complement to mouse models, especially for cell-autonomous pathways like the circadian clock. In this study, we quantitatively assessed the use of CRISPR to create cell models in mouse embryonic fibroblasts (MEFs) as compared to mouse models. We generated two point mutations in the clock genes Per1 and Per2 in mice and in MEFs using the same sgRNAs and repair templates for HDR and quantified the frequency of the mutations by digital PCR. The frequency was about an order of magnitude higher in mouse zygotes compared to that in MEFs. However, the mutation frequency in MEFs was still high enough for clonal isolation by simple screening of a few dozen individual cells. The Per mutant cells that we generated provide important new insights into the role of the PAS domain in regulating PER phosphorylation, a key aspect of the circadian clock mechanism. Quantification of the mutation frequency in bulk MEF populations provides a valuable basis for optimizing CRISPR protocols and time/resource planning for generating cell models for further studies.

Identification of a serodiagnostic antigen, legumain, by immunoproteomic analysis of excretory-secretory products of Clonorchis sinensis adult worms.

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.

An Excel Macro for Determining Allelic and Sequence Types of Bacterial Clones in Multilocus Sequence Typing.

BACKGROUND: Multilocus sequence typing (MLST) was designed to overcome the low discriminatory power and poor reproducibility of previous molecular typing schemes, and it is useful for inter-laboratory, inter-regional, and inter-national comparison of pathogenic clones. MLST includes labor-intensive sequencing processes and meticulous allelic/sequence type (ST) determination processes, often prone to error. We developed a free automated MLST determination program (MLST typer) based on the Visual Basic for Applications macro, which runs on Microsoft Excel. METHODS: MLST typer imports sequence data in the FASTA format, converts reverse complement counterparts of the reverse sequences, assembles forward and reverse-complement converted sequences, and returns allelic numbers for each gene and ST of each isolate. To evaluate the performance of MLST typer, we tested the sequence data from 200 clinical isolates, each consisting of seven housekeeping gene sequences, with a total of 1,400 allelic number determinations. The results were compared with manual assessment. RESULTS: MLST typer comprises three worksheets: the Main page, Result page, and Summary page. The Main page console operates the process according to user-specified parameters. The Result and Summary pages provide the allelic type and ST determinations. It took approximately 12 minutes to analyze the sequence data from 200 clinical isolates. Compared with manual assessment, the rate of correct identification was 97.2% (1,361/1,400). CONCLUSIONS: MLST typer can be widely used for epidemiological studies owing to its thoroughness in repetitive functions, good compatibility with FASTA type data files, and easy-to-understand outputs for allelic and ST determinations.

Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9.

CRISPR-Cas9 is a powerful gene editing technique that can induce mutations in a target gene of interest in almost any mammalian cell line. However, its practicality can be limited if target cell lines are difficult to transfect and do not proliferate. In the current study, we have developed a streamlined approach for CRISPR-based gene knockouts with three key advantages, which allows phenotypic assay of gene knockouts without clonal selection and expansion. First, it integrates into a single, all-in-one vector transgenes for Cas9, sgRNA, and a fluorescence marker. Second, we used the Gateway system to rapidly clone specific sgRNAs into the all-in-one vector through PCR and in vitro recombination, without conventional enzyme digestion and ligation. Third, it uses adenovirus for the capacity to package the all-in-one vector, and for its high efficiency of transduction. We tested the all-in-one adenoviral CRISPR-Cas9 in a circadian clock model cell line U2OS, and demonstrated that essential clock genes such as Bmal1 and Per1 were knocked out so efficiently that functional assays could be performed from the heterogenic population without any clonal selection and expansion. This streamlined approach may prove invaluable for rapid functional assays of candidate genes in diverse biological pathways, including the circadian clock.

The effect of protein expression of Streptococcus pneumoniae by blood.

During infection, the common respiratory tract pathogen Streptococcus pneumoniae encounters several environmental conditions, such as upper respiratory tract, lung tissue, and blood stream, etc. In this study, we examined the effects of blood on S. pneumoniae protein expression using a combination of highly sensitive 2-dimensional electrophoresis (DE) and MALDI-TOF MS and/or LC/ESI-MS/MS. A comparison of expression profiles between the growth in THY medium and THY supplemented with blood allowed us to identify 7 spots, which increased or decreased two times or more compared with the control group: tyrosyl-tRNA synthetase, lactate oxidase, glutamyl-aminopeptidase, L-lactate dehydrogenase, cysteine synthase, ribose-phosphate pyrophosphokinase, and orotate phosphoribosyltransferase. This global approach can provide a better understanding of S. pneumoniae adaptation to its human host and a clue for its pathogenicity.

Proteomic analysis of protein expression in Streptococcus pneumoniae in response to temperature shift.

From its initial colonization to causation of disease, Streptococcus pneumoniae has evolved strategies to cope with a number of stressful in vivo environmental conditions. In order to analyze a global view of this organism's response to heat shock, we established a 2-D electrophoresis proteome map of the S. pneumoniae D39 soluble proteins under in vitro culture conditions and performed the comparative proteome analysis to a 37 to 42 degrees temperature up-shift in S. pneumoniae. When the temperature of an exponentially growing S. pneumoniae D39 culture was raised to 42 degrees , the expression level of 25 proteins showed changes when compared to the control. Among these 25 proteins, 12 were identified by MALDI-TOF and LC-coupled ESI MS/MS. The identified proteins were shown to be involved in the general stress response, energy metabolism, nucleotide biosynthesis pathways, and purine metabolism. These results provide clues for understanding the mechanism of adaptation to heat shock by S. pneumoniae and may facilitate the assessment of a possible role for these proteins in the physiology and pathogenesis of this pathogen.

Outbreak of respiratory tract infections on an islet in Korea: possible Chlamydia pneumoniae infection.

In March 2004, we experienced an outbreak of Chlamydia pneumoniae infection on an islet of Korea. In order to assess the significance of the epidemic, we performed a mass examination of 137 students (7-16 years old; male, 69; female, 58) at a school. The examination consisted of a questionnaire inquiring about respiratory symptoms, a serum antibody test for C. pneumoniae using a microimmunofluorescence (MIF) method and enzyme-linked immunosorbent assay (ELISA), and nasopharyngeal swab tests to detect of the organism by specific PCR and cell culture. The results demonstrated that 72 (58.3%) of the students had respiratory symptoms such as rhinorrhea, a sore throat, and/or cough or fever. The PCR positivity of acute-phase patients was 63% (12/19) and PCR positivity using the culture sample was 94% (18/19). However, the existence of the organism was not confirmed fluorescein isothiocyanate (FITC). ELISA, one of the serological methods utilized, demonstrated, in the same patients, 48% (13/27) positive IgM antibodies at the acute phase of the outbreak, and 16% (3/19) positive IgM antibodies during the convalescent phase. The index value (ID) 3.0 for single-sera IgG was 19% (5/27) and that for IgA was 4% (1/27) at the acute phase; the corresponding percentages in the convalescent phase were 11% (2/19) and 5% (1/19), respectively. However, as regards paired sera, no patient demonstrated a 1.35 ELISA ID value at 2 weeks, or an increased value of 1.0 at 8 weeks after the onset of the outbreak. In the MIF experiment, the percent positivity of unpaired IgM from the acute phase was 58% (11/19). At convalescent phase, this percentage was 47% (9/19); however, the positivity of paired serum IgG was 26% (5/19). In the same sample, the percentage of positive cases demonstrated by both ELISA and MIF approaches for single IgM was 37% (7/19) at the acute phase and 11% (2/19) at the convalescent phase. We were unable to isolate C. pneumoniae by cell culture, but we did obtain sufficient serological and PCR data to consider C. pneumoniae as the causative agent of the outbreak. Meaningful results were acquired in terms of serology, and were compared to the healthy population in Korea. Although it remains necessary to investigate the possibility of co-infection and to determine whether or not this outbreak coincides with the prevalence of influenza, it was unequivocally concluded that this outbreak of C. pneumoniae infection has occurred on an islet of Korea.

Case of childhood granulomatous periorificial dermatitis in a Korean boy treated by oral erythromycin.

Childhood granulomatous periorificial dermatitis (CGPD) is a disease presenting most commonly in prepubertal children as yellow-brown papules limited to the perioral, perinasal and periocular regions. The condition is benign, self-limiting and is not associated with systemic involvement. We herein report a case of an 11-year-old Korean boy with multiple, asymptomatic, monomorphic, red-to-yellow-colored papular eruptions on the perioral areas of 7-month duration. Histopathological examination revealed upper dermal and perifollicular granulomatous infiltrate. After using oral erythromycin 500 mg daily for 1 year, the condition resolved completely without leaving a scar.

Botulinum neurotoxin type A induces TLR2-mediated inflammatory responses in macrophages.

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.

Establishing quality control ranges for antimicrobial susceptibility testing of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus: a cornerstone to develop reference strains for Korean clinical microbiology laboratories.

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.

Changes in serotype prevalence and antimicrobial resistance among invasive Streptococcus pneumoniae isolates in Korea, 1996-2008.

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 <5 years old and in adults ≥65 years old between 1996-1999 and 2007-2008. An increased prevalence of non-PCV7 serotypes in these age groups was primarily attributable to an increase in serotypes 3, 6A and 19A. Most invasive 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.