Rapid Emergence and Clonal Dissemination of CTX-M-15-Producing Salmonella enterica Serotype Virchow, South Korea.

The prevalence of cefotaxime-resistant Salmonella enterica serotype Virchow has dramatically increased in South Korea since the first isolation in 2011. Of 68 isolates collected over 10 years, 28 cefotaxime-resistant isolates harbored the bla(CTX-M-15) extended-spectrum ß-lactamase gene and were closely related genetically, demonstrating the clonal dissemination of CTX-M-15-producing Salmonella Virchow in South Korea.

Consecutive Outbreaks of Enterotoxigenic Escherichia coli O6 in Schools in South Korea Caused by Contamination of Fermented Vegetable Kimchi.

BACKGROUND: Two outbreaks of gastroenteritis occurred in South Korea, affecting a middle school in the Jeollanam-do province in 2013 (Outbreak 1) and 10 schools in the Incheon province in 2014 (Outbreak 2). We investigated the outbreaks to identify the pathogen and mode of transmission. METHODS: A retrospective cohort study was conducted in the Outbreak 1; and case-control studies were performed for the Outbreak 2. Samples from students, environments, and preserved food items were collected and pulsed-field gel electrophoresis (PFGE) was conducted to identify strains of pathogen. RESULTS: We identified 167 and 1022 students who met the case definition (≥3 loose stools in any 24-h period) in the Outbreaks 1 and 2, respectively. The consumption of cabbage kimchi and young radish kimchi were significantly associated with the illness. Adjusted odds ratios of kimchi were 2.62-11.74. In the Outbreak 1, cabbage kimchi was made and consumed in the school restaurant and in the Outbreak 2, young radish kimchi was supplied by food company X and distributed to all the 10 schools in the Incheon province. Enterotoxigenic Escherichia coli (ETEC) O6 was isolated from fecal samples in 375 cases (33.9%) and from kimchi samples. PFGE patterns of the outbreak strains isolated from cases and food were indistinguishable in each outbreak. CONCLUSION: The suspected food vehicle in these two consecutive outbreaks was kimchi contaminated with ETEC O6. We recommend continued monitoring and stricter sanitation requirements for the food supply process in Korea, especially in relation to kimchi.

Outbreak of Ciprofloxacin-Resistant Shigella sonnei Associated with Travel to Vietnam, Republic of Korea.

We investigated an October 2014 outbreak of illness caused by Shigella sonnei in a daycare center in the Republic of Korea (South Korea). The outbreak strain was resistant to extended-spectrum cephalosporins and fluoroquinolones and was traced to a child who had traveled to Vietnam. Improved hygiene and infection control practices are needed for prevention of shigellosis.

Comparative proteomic label-free analysis of Campylobacter jejuni NCTC 11168 cultured with porcine mucin.

Campylobacter jejuni is a major gastrointestinal pathogen in humans. Poultry is a primary reservoir for C. jejuni, and C. jejuni appears to be highly adapted to the gastrointestinal tracts of avian species. We determined the protein expression profiles of C. jejuni NCTC 11168 cultured in medium containing porcine mucin. Differentially expressed proteins in the presence and absence of porcine mucin were identified using the label-free method. We identified 52 proteins with expression that was either upregulated (32 proteins) or downregulated (20 proteins) by porcine mucin. These proteins are involved in diverse cellular functions, such as motility, cell wall synthesis, iron transport, energy production, and amino acid metabolism. In particular, the upregulated proteins were involved in chemotaxis (CheV and CetA), motility (FlaA), colonization and adherence (CadF, FrdA, CfrA, MapA, and HydA), and stress tolerance (TrxB and ClpB). These results suggest that C. jejuni changes its protein expression in response to porcine mucin and that this change in expression may contribute to host adaptation of C. jejuni NCTC 11168.

Improved Immunogenicity of the Inactivated F Genotype Mumps Vaccine against Diverse Circulating Mumps Viruses in Mice.

Mumps is an acute infectious disease caused by the mumps virus (MuV). Despite high global vaccination coverage, mumps outbreaks continue to occur, even in vaccinated populations. Therefore, we aimed to identify candidate vaccines that can induce an immunogenic response against diverse MuV genotypes with greater efficacy than the currently available options. Vaccine candidates were sourced using formalin-inactivated viral strains. The inactivated vaccines were administered to BALB/c mice (through a primer and booster dose administered after a three-week interval). We tested the neutralizing antibodies of the candidate vaccines against various MuV genotypes to determine their overall efficacy. The formalin-inactivated F genotype vaccine was found to have higher cross-neutralizing titers against genotypes F, H, and G as well as significant Th1 cytokines responses, IFN-γ, TNF-α, and IL-2 than the Jeryl Lynn (JL) vaccine. Our findings suggest that the inactivated F genotype mumps vaccine has higher immunogenicity than the JL vaccine against diverse circulating MuVs.

Comparison of plaque reduction and focus reduction neutralization tests for the measurement of neutralizing antibody titers against japanese encephalitis virus.

Japanese encephalitis is prevalent throughout the temperate and tropical regions of Asia and is caused by the Japanese encephalitis virus (JEV), a mosquito-borne viral pathogen. The plaque reduction neutralization test (PRNT) is currently recommended as the gold standard test for detecting human antibodies against JEV. The plaque assay is the most widely used method for detecting infectious virions and involves counting discrete plaques in cells. However, it is time-consuming, and results can be subjective (owing to analyst variability during manual plaque counting). The focus reduction neutralization test (FRNT), which is based on an immuno-colorimetric assay, can be used to automatically count foci formed by the JEV. Here, we compared the efficacy of PRNT and FRNT in measuring the neutralizing antibody titers using 102 serum samples from vaccinated and unvaccinated individuals. We observed positive correlations between these neutralization assays against the Nakayama and Beijing strains (R2 = 0.98 and 0.77, respectively). Thus, FRNT may be preferable to PRNT for evaluating the efficacy of JEV vaccines in large-scale serological studies.

Immunogenicity of candidate SARS-CoV-2 DNA vaccines based on the spike protein.

Coronavirus disease 2019 caused by the novel human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently a major threat to public health worldwide. To deal with the needs of vaccine, we developed four DNA vaccine candidates against SARS-CoV-2, based on the full-length spike (S) or truncated S protein. Following mice vaccination, we measured T-cell response and antigen-specific neutralizing antibody (NAb) titer. All four candidates induced humoral immune responses, including elevated levels of total IgG and NAbs, and cell-mediated immune responses, including multiple cytokine expression. However, the full-length S DNA vaccine enhanced the immune responses most significantly. We then evaluated its appropriate antigen dose and vaccination schedule. Although all immunized groups showed higher immune response than the control group, inoculation with 50 µg antigen led to the highest NAb titer. Immunity was significantly increased after the third inoculation. Thus, the full-length S DNA vaccine can potentially prevent SARS-CoV-2 infection.

Molecular characterization of methicillin-resistant Staphylococcus aureus isolates in Korea.

We used several molecular typing methods to analyze 196 methicillin-resistant Staphylococcus aureus (MRSA) and 139 methicillin-susceptible S. aureus (MSSA) isolates collected between 1996 and 2005. The sequence type 72 MRSA has increased in frequency in the community in the Republic of Korea and in hospitals in recent years.

Optimization of Zika DNA vaccine by delivery systems.

In our previous study, we designed and evaluated the efficacy of six DNA vaccine candidates based on the E protein of Zika virus (ZIKV). To optimize the DNA vaccine, we inoculated C57BL/6 and IFNAR1- mice with the vaccine candidate expressing tandem repeated ZIKV envelope domain III (ED III × 3) doses; 50 µg by intramuscular (IM), jet injection (JET), or electroporation (EP) routes. Results showed that vaccination by all routes induced humoral and cellular immunity. Among them, EP induced robust ZIKV E specific-total IgG and neutralizing antibodies, as well as T cell responses. Additionally, EP showed superior protective efficacy against the ZIKV Brazil strain compared to the IM and JET routes. Finally, in the dose optimization test of EP route, cellular immunity of 50 µg was induced a significant level than other dose groups. These results showed that the EP delivery system enhanced the potential immunogenicity and protective efficacy of DNA vaccines.

Cross-neutralization between vaccine and circulating wild-type mumps viruses in Korea.

Mumps is a contagious disease caused by the mumps virus. It can be prevented using mumps vaccines, administered as a measles-mumps-rubella (MMR) vaccine. For first and second dose immunization, children aged 12-15 months and 4-6 years have been administered this vaccine since 1997 in Korea. Nevertheless, mumps outbreaks still occur in vaccinated populations worldwide. Hence, immunity against these diseases may be attenuated, or there are antigenic differences between currently available vaccine strains and circulating wild-type viruses. After the introduction of national immunization programs in Korea, mumps cases became sporadic. Viral genotypes F, H, and I have emerged since 1998 whereas the vaccine strains belong to genotype A. Here, we compared the amino acid sequences of the haemagglutinin-neuraminidase (HN) gene from wild-type viruses and the mumps vaccine and measured the cross-neutralization titers between them. We selected the F, H, and I wild-type mumps strains circulating in Korea from 1998 to 2016 and analyzed changes in the amino acid sequence of the protein encoded by the HN gene. We measured mumps virus-specific IgG and rapid focus reduction neutralization test (FRNT) titers in Korean isolates and sera obtained from 50 children aged 1-2 years who had been administered a single dose of MMR vaccine. Analysis of the HN protein sequences disclosed no changes in the glycosylation sites but did reveal 4-5 differences between the Korean isolates and the genotype A vaccine strain in terms of the neutralizing epitope sites on their HN proteins. Post-vaccination FRNT titers were significantly lower against genotypes F, H, and I than they were against genotype A. This finding highlights the possibility of a recurrence of mumps outbreaks in vaccinated populations depending on the degree of genetic conservation of the HN gene. Further research into this issue is needed to prevent the resurgence of mumps.

Development of an attenuated smallpox vaccine candidate: The KVAC103 strain.

Smallpox, a disease caused by the variola virus, is one of the most dangerous diseases and had killed numerous people before it was eradicated in 1980. However, smallpox has emerged as the most threatening bio-terrorism agent; as the first- and second-generation smallpox vaccines have been controversial and have caused severe adverse reactions, new demands for safe smallpox vaccines have been raised and some attenuated smallpox vaccines have been developed. We have developed a cell culture-based highly attenuated third-generation smallpox vaccine candidate KVAC103 strain by 103 serial passages of the Lancy-Vaxina strain derived from the Lister in Vero cells. Several clones were selected, taking into consideration their shape, size, and growth rate in mammalian cells. The clones were then inoculated intracerebrally in suckling mice to test for neurovirulence by observing survival. Protective immune responses in adult mice were examined by measuring the levels of neutralization antibodies and IFN-γ expression. Among several clones, clone 7 was considered the best alternative candidate because there was no mortality in suckling mice against a lethal challenge. In addition, enhanced neutralizing antibodies and T-cell mediated IFN-γ production were observed in clone 7-immunized mice. Clone 7 was named "KVAC103" and was used for the skin toxicity test and full-genome analysis. KVAC103-inoculated rabbits showed reduced skin lesions compared to those inoculated with the Lister strain, Lancy-Vaxina. A whole genome analysis of KVAC103 revealed two major deleted regions that might contribute to the reduced virulence of KVAC103 compared to the Lister strain. Phylogenetic inference supported the close relationship with the Lister strain. Collectively, our data demonstrate that KVAC103 holds promise for use as a third-generation smallpox vaccine strain due to its enhanced safety and efficacy.

Characterization of two newly identified genes, vgaD and vatH, [corrected] conferring resistance to streptogramin A in Enterococcus faecium.

We characterized two new streptogramin A resistance genes from quinupristin-dalfopristin-resistant Enterococcus faecium JS79, which was selected from 79 E. faecium isolates lacking known genes encoding streptogramin A acetyltransferase. A 5,650-bp fragment of HindIII-digested plasmid DNA from E. faecium JS79 was cloned and sequenced. The fragment contained two open reading frames carrying resistance genes related to streptogramin A, namely, genes for an acetyltransferase and an ATP efflux pump. The first open reading frame comprised 648 bp encoding 216 amino acids with a predicted left-handed parallel ß-helix domain structure; this new gene was designated vatH. [corrected] The second open reading frame consisted of 1,575 bp encoding 525 amino acids with two predicted ATPase binding cassette transporters comprised of Walker A, Walker B, and LSSG motifs; this gene was designated vgaD. vgaD is located 65 bp upstream from vatH, [corrected] was detected together with vatH [corrected] in 12 of 179 quinupristin-dalfopristin-resistant E. faecium isolates, and was located on the same plasmid. Also, the 5.6-kb HindIII-digested fragment which was observed in JS79 was detected in nine vgaD- and vatH-containing [corrected] E. faecium isolates by Southern hybridization. Therefore, it was expected that these two genes were strongly correlated with each other and that they may be composed of a transposon. Importantly, vgaD is the first identified ABC transporter conferring resistance to streptogramin A in E. faecium. Pulsed-field gel electrophoresis patterns and sequence types of vgaD- and vatH-containing [corrected] E. faecium isolates differed for isolates from humans and nonhumans.

Various penA mutations together with mtrR, porB and ponA mutations in Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime or ceftriaxone.

OBJECTIVES: To examine mutations within the penA, mtrR, porB, ponA and pilQ genes of Neisseria gonorrhoeae to determine their contribution to cephalosporin resistance. METHODS: A total of 46 N. gonorrhoeae isolates with reduced susceptibility to cefixime or ceftriaxone (MICs > or = 0.12 mg/L) and two susceptible isolates were selected. The full sequence of penA and partial sequences previously reported as hot mutation sites of the other genes were analysed. Genotyping by N. gonorrhoeae multiantigen sequence typing (NG-MAST) was also performed. RESULTS: A mosaic penicillin-binding protein 2 (PBP 2) was found in a single isolate that exhibited the highest cefixime MIC (0.5 mg/L). The majority of the isolates with reduced susceptibility to cephalosporins contained non-mosaic PBP 2 sequences, of which PBP 2 pattern XIII was most common (28/46). All isolates with reduced susceptibility to cephalosporins also had mtrR and porB mutations. Two susceptible isolates had the PBP 2 pattern XIV and an incomplete MtrR protein, which was a new mutation. Isolates with identical PBP 2 patterns comprised multiple NG-MAST sequence types. CONCLUSIONS: Reduced susceptibility of N. gonorrhoeae to ceftriaxone and cefixime was associated with diverse penA mutations, particularly PBP 2 pattern XIII containing an Ala-501-->Val substitution, together with mtrR and porB mutations. The existence of only one strain having the mosaic penA sequence indicated that ceftriaxone and cefixime resistance in Korea is mostly not associated with a mosaic penA sequence. Highly heterogeneous NG-MAST sequence types excluded the clonal expansion of a particular subtype.

Enhanced effect of modified Zika virus E antigen on the immunogenicity of DNA vaccine.

It has been reported worldwide that the Zika virus (ZIKV) could be transmitted through placentas and sexual contact. ZIKV can also cause Guillain-Barre syndrome, microcephaly and neurological abnormalities. However, there are no approved vaccines available. We constructed six DNA vaccine candidates and tested the immunogenicity. Tandem repeated envelope domain Ⅲ (ED Ⅲ × 3) induced highly total IgG and neutralization antibody, as well as CD8+ T cell responses. Also, stem region-removed envelope (E ΔSTEM) elicited a robust production of IFN-γ in mice. To examine in vivo protection, we used mice treated with an IFNAR1 blocking antibody before and after the challenge. Vaccination with the two candidates led to a decline in the level of viral RNAs in organs. Moreover, the sera from the vaccinated mice did not enhance the infection of Dengue virus in K562 cells. These findings suggest the potential for the development of a novel ZIKV DNA vaccine.

Investigation of Klebsiella pneumoniae isolates producing SHV-12 and SHV-11 beta-lactamases in Korean hospitals.

Of 143 clinical isolates of Klebsiella pneumoniae collected from Korean non-tertiary hospitals, 24 (16.8%) showed an extended-spectrum beta-lactamase-positive phenotype. PCR and sequence analysis revealed the presence of TEM-116 (n=13), CTX-M-3 (n=5), CTX-M-14 (n=2), CTX-M-15 (n=3), and SHV-12 (n=16). Each of the 24 isolates encoded more than one beta-lactamase, and seven isolates (29%) harbored two different SHV-type beta-lactamase genes (blaSHV-11 and blaSHV-12) bounded by insertion sequence IS26 in a single transferable plasmid.

Efficient expression of enterovirus 71 based on virus-like particles vaccine.

Enterovirus (EV) 71 is the main pathogen associated with hand-foot-mouth disease (HFMD) and can lead to the disease with severe mortality in children. Since 2009, in the Republic of Korea, an outbreak of EV71 C4a infection with neurologic involvement emerged, where in HFMD involvement was identified and central nervous system complications were reported. In this study, EV71 C4a virus-like particles (VLPs) produced by recombinant technology were generated in a baculovirus expression system. To improve the production yield, EV71 VLP was constructed using the dual promoter system baculovirus P1 and 3CD (baculo-P1-3CD), which harbored both the structural protein-encoding P1 region under the control of the polyhedron promoter and the 3CD protease gene under the regulation of the CMV-IE, lef3, gp41, or chitinase promoters to augment the level of gene transcription. Efficient VLP expression was demonstrated through optimization of incubation time and insect cell type. In addition, to evaluate the potential of VLP as a vaccine candidate, we tested the neutralizing antibodies and total anti-EV71 IgG from the purified EV71 C4a VLP serum. The recombinant EV71 VLP exhibited the morphology of self-assembled VLP, as determined by electron microscopy. Use of baculo-P1-3CD-gp41 led to a high yield (11.3mg/L < 40kDa) of VLPs in High-FiveTM cells at 3 days post-infection. Furthermore, the potential of VLP as a vaccine was evaluated through the neutralizing ability elicited by the purified EV71 VLP after immunization of BALB/c mice, which was shown to induce potent and long-lasting humoral immune responses as evidenced by the cross-neutralization titer. Our results could be used to expedite the developmental process for vaccines under clinical trials and to ensure manufacturing consistency for licensing requirements.

Complete genome sequence of Neisseria gonorrhoeae NCCP11945.

Neisseria gonorrhoeae is an obligate human pathogen that is the etiological agent of gonorrhea. We explored variations in the genes of a multidrug-resistant N. gonorrhoeae isolate from a Korean patient in an effort to understand the prevalence, antibiotic resistance, and importance of horizontal gene transfer within this important, naturally competent organism. Here, we report the complete annotated genome sequence of N. gonorrhoeae strain NCCP11945.

Five-year report of national surveillance of antimicrobial resistance in Pseudomonas aeruginosa isolated from non-tertiary care hospitals in Korea (2002-2006).

A nationwide surveillance of the antimicrobial resistance of Pseudomonas aeruginosa isolates from non-tertiary care hospitals was conducted in Korea from 2002 to 2006. Resistance to almost all antimicrobial agents decreased significantly from 2003 (P < 0.01). Resistance rates to the major antipseudomonal agents, ceftazidime, imipenem, meropenem, and aztreonam, were 18.8%, 20.5%, 18.7%, and 19.7%, respectively, in 2003. However, they had all decreased to below 10% in 2006. The proportion of multidrug-resistant isolates that were resistant to at least 3 of 5 major antipseudomonal agent decreased from 33.5% in 2003 to 23.1% in 2006 (P < 0.05). In this study, we found a decreasing trend in resistance rates and low resistance rates in P. aeruginosa from non-tertiary care hospitals compared with those from general hospitals, including tertiary care hospitals, in Korea. Our data provide valuable information for the selection of reliable empiric therapies for P. aeruginosa infections in non-tertiary care hospital patients, including outpatients.

The immunogenicity and protection effect of an inactivated coxsackievirus A6, A10, and A16 vaccine against hand, foot, and mouth disease.

Coxsackievirus belongs to the Enterovirus genus of the Picornaviridae family and is one of the major pathogens associated with human hand, foot, and mouth disease (HFMD). Historically, outbreaks of HFMD have mainly been caused by enterovirus 71 and coxsackievirus A16. Recently, coxsackieviruses A6 and A10 have been associated with increased occurrences of sporadic HFMD cases and outbreak events globally. In this study, the immunogenicity of coxsackieviruses A6, A10, and A16 (CA6, CA10, and CA16), which were inactivated by formalin or ß-propiolactone (BPL) under different conditions, was evaluated as multivalent vaccine candidates. CA6 induced similar immune responses with both inactivation methods, and the immune efficacy of CA10 and CA16 was better following inactivation with BPL than with formalin. There was no sufficient cross-reactivity or cross-protectivity against heterologous strains in groups vaccinated with the BPL-inactivated (BI) monovalent vaccine. Sufficient neutralizing antibody and cell-mediated immune responses were induced in the BI-trivalent vaccinated group. These findings suggest that BI-CA6, CA10, and CA16 are potential multivalent vaccine candidates and that a multivalent vaccine is needed to control HFMD. The coxsackievirus multivalent vaccine could be useful for the development of effective HFMD vaccines.

Estimates of short- and long-term incubation periods of Plasmodium vivax malaria in the Republic of Korea.

With the current epidemic of vivax malaria closely associated with the demilitarised zone along the border between North and South Korea, it has been suggested that the incubation period tends, in part, to be prolonged. Based on the detailed travel history of cases from 2000 to 2003 who reside in non-malarious areas, statistical estimates of the incubation periods were obtained. The data suggest that cases fall into two categories with short- and long-term incubation periods, respectively. Of 416 cases with available information, 72 and 79 successfully met our criteria for inferring the durations of short- and long-term incubation periods. The mean short- and long-term incubation periods were estimated to be 26.6 days (95% CI 21.0-32.2) and 48.2 weeks (95% CI 46.8-49.5), respectively. The maximum likelihood method was used to fit gamma and normal distributions to the short- and long-term incubation periods, assisting prediction of the frequency distribution of the overall incubation period, which exhibited a bimodal pattern. We postulate that the observed distribution reflects adaptation of the parasite to the seasonal population dynamics of the vector, Anopheles sinensis, ensuring continued transmission of vivax malaria in this temperate zone.