Search

Additional file 3.

Additional file 2.

Additional file 1.

Background Anthrax and smallpox are high-risk infectious diseases, and considered as potential agents for bioterrorism. To develop an effective countermeasure for these diseases, we constructed a bivalent vaccine against both anthrax and smallpox by integrating a gene encoding protective antigen (PA) of Bacillus anthracis to the genome of the attenuated vaccinia virus strain, KVAC103. Results Immunization with this bivalent vaccine induced antibodies against both PA and vaccinia virus in a mouse model. We also observed that the efficacy of this vaccine can be enhanced by combined immunization with immunoadjuvant-expressing KVAC103. Mouse groups co-immunized with PA-expressing KVAC103 and either interleukin-15 (IL-15) or cholera toxin subunit A (CTA1)-expressing KVAC103 showed increased anti-PA IgG titer and survival rate against B. anthracis spore challenge compared to the group immunized with PA-expressing KVAC103 alone. Conclusions We demonstrated that the attenuated smallpox vaccine KVAC103 is an available platform for a multivalent vaccine and co-immunization of immunoadjuvants can improve vaccine performance.

Sequence type (ST) 33 of Shiga toxin-producing Escherichia coli (STEC) strain O91:H14 has been proposed as a potential domestic clone of STEC in Korea because of its high prevalence among human patients with mild diarrhea or asymptomatic carriers. Herein, the clonal diversity of 17 STEC O91:H14 isolates of ST33 during 2003 to 2014 was analyzed by pulsed-field gel electrophoresis, including 14 isolates from human patients and 3 from retail meats. Their virulence characteristics, acid resistance, and antimicrobial susceptibility were also determined. Our results showed that all isolates were clustered mainly into three different pulsotypes and were likely low pathogenic without antimicrobial resistance.

We report here the draft genome sequence of Burkholderia pseudomallei H0901. This strain was isolated in 2003 from the first melioidosis patient in South Korea.

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.

We report here the draft genome sequences of four vancomycin-intermediate Staphylococcus aureus (VISA) strains from South Korean hospitals participating in a nationwide laboratory surveillance program for vancomycin-intermediate and vancomycin-resistant Staphylococcus aureus . All strains harbor mutations in the walKR , graSR , and/or rpoB genes that are known frequently mutated determinants of VISA.

SUMMARYThis study investigated the carriage of antimicrobial resistantHaemophilus influenzaein 582 healthy children attending kindergarten or elementary school at four intervals over a 9-month period in Seoul, Korea. Diverse colonization patterns and a lower level of long-term persistent carriage byH. influenzaestatus were evident in this study. ColonizingH. influenzaeisolates showed a high rate of resistance to β-lactams including ampicillin (51·9%), cefaclor (52·1%), and amoxicillin/clavulanate (16·3%). Based on the ampicillin resistance mechanism,H. influenzaeisolates were categorized as β-lactamase-negative, ampicillin-susceptible (BLNAS) (48·1%), β-lactamase-positive, ampicillin-resistant (BLPAR) (22·6%), β-lactamase-negative, ampicillin-resistant (BLNAR) (22·8%), and β-lactamase-positive, amoxicillin/clavulanate-resistant (BLPACR) strains (6·5%). This study provides the first evidence of a high prevalence (22·8%) of BLNAR strains ofH. influenzaenasal carriage in healthy children attending kindergarten or the first 2 years of elementary school in Korea. The high carriage of these resistant strains in overcrowded urban settings may create reservoirs for development ofH. influenzae-resistant strains.

Objectives To confirm genotype diversities of clinical isolates of Bordetella pertussis and to evaluate the risk of pertussis outbreak in Korea. Methods Seven housekeeping genes and 10 antigenic determinant genes from clinical B. pertussis isolates were analyzed by Multilocus sequence typing (MLST). Results More variant pattern was observed in antigenic determinant genes. Especially, PtxS1 gene was the most variant gene; five genotypes were observed from eight global genotypes. In the bacterial type, the number of observed sequence types in the isolates was seven and the most frequent form was type 1 (79.6%). This major sequence type also showed a time-dependent transition pattern. Older isolates (1968 and 1975) showed type 1 and 6 in housekeeping genes and antigenic determinant genes, respectively. However, these were changed to type 2 and 1 in isolates 1999–2008. This transition was mainly attributed to genotype change of PtxS1 and Fim3 gene; the tendency of genotype change was to avoid vaccine-derived genotype. In addition, there was second transition in 2009. In this period, only the sequence type of antigenic determinant genes was changed to type 2. Based Upon Related Sequence Types (BURST) analysis confirmed that there were two clonal complexes (ACCI and ACCII) in the Korean isolates. Moreover, the recently increased sequence type was revealed as AST2 derived from AST 3 in ACCI. Conclusions Genotype changes in Korean distributing strains are still progressing and there was a specific driving force in antigenic determinant genes. Therefore continuous surveillance of genotype change of the distributing strains should be performed to confirm interrelationship of genotype change with vaccine immunity.

Sequence type (ST) 33 of Shiga toxin-producing Escherichia coli (STEC) strain O91:H14 has been proposed as a potential domestic clone of STEC in Korea because of its high prevalence among human patients with mild diarrhea or asymptomatic carriers. Herein, the clonal diversity of 17 STEC O91:H14 isolates of ST33 during 2003 to 2014 was analyzed by pulsed-field gel electrophoresis, including 14 isolates from human patients and 3 from retail meats. Their virulence characteristics, acid resistance, and antimicrobial susceptibility were also determined. Our results showed that all isolates were clustered mainly into three different pulsotypes and were likely low pathogenic without antimicrobial resistance.

Antimicrobial susceptibility patterns and β-lactam resistance mechanisms of 544 Haemophilus influenzae isolates through the nationwide Acute Respiratory Infections Surveillance (ARIS) network in Korea during 2005 and 2006 were determined. Resistance to ampicillin was 58.5%, followed by resistance to cefuroxime (23.3%), clarithromycin (18.7%), cefaclor (17.0%), amoxicillin-clavulanate (10.4%), and chloramphenicol (8.1%). Levofloxacin and cefotaxime were the most active agents tested in this study. β-Lactamase production (52.4%) was the main mechanism of ampicillin resistance, affecting 96.1% of TEM-1-type β-lactamase. According to their β-lactam resistance mechanisms, all isolates were classified into the following groups: β-lactamase-negative, ampicillin-sensitive (BLNAS) strains ( n = 224; 41.5%); β-lactamase-positive, ampicillin-resistant (BLPAR) strains ( n = 255; 47.2%); β-lactamase-negative, ampicillin-resistant (BLNAR) strains ( n = 33; 6.1%); and β-lactamase-positive, amoxicillin-clavulanate-resistant (BLPACR) strains ( n = 28; 5.2%). Among the BLNAR and BLPACR strains, there were various patterns of multiple-amino-acid substitutions in penicillin-binding protein 3. Particularly, among BLNAR, group III isolates, which had three simultaneous substitutions (Met377Ile, Ser385Thr, and Leu389Phe), were identified for the first time in Korea. Three group III strains displayed the highest MIC of cefotaxime (1 to 2 μg/ml). The results indicate the importance of monitoring a changing situation pertaining to the increase and spread of BLNAR and BLPACR strains of H. influenzae for appropriate antibiotic therapy for patients with respiratory tract infections in Korea.

Current human anthrax vaccines available in the United States and Europe consist of alum-precipitated supernatant material from cultures of a toxigenic, nonencapsulated strain of Bacillus anthracis. The major component of human anthrax vaccine that confers protection is protective antigen (PA). A second-generation human vaccine using the recombinant PA (rPA) is being developed. In this study, to prevent the toxicity and the degradation of the native rPA by proteases, we constructed two PA variants, delPA (163–168) and delPA (313–314), that lack trypsin (S 163 –R 164 –K 165 –K 166 –R 167 –S 168 ) or chymotrypsin cleavage sequence (F 313 –F 314 ), respectively. These proteins were expressed in Bacillus brevis 47-5Q. The delPAs were fractionated from the culture supernatant of B. brevis by ammonium sulfate at 70% saturation, followed by anion exchange chromatography on a Hitrap Q, Hiload 16/60 superdex 200 gel filtration column and phenyl sepharose hydrophobic interaction column. In accordance with previous reports, both delPA proteins combined with lethal factor protein did not show any cytotoxicity on J774A.1 cells. The delPA (163– 168) and delPA (313–314) formulated either in Rehydragel HPA or MPL-TDM-CWS (Ribi-Trimix), elicited a comparable amount of anti-PA and neutralizing antibodies to those of native rPA in guinea pigs, and confers full protection of guinea pigs from 50 · LD50 of fully virulent B. anthracis spore challenges. Ribi-Trimix was significantly more effective in inducing anti-PA and neutralizing antibodies than Rehydragel HPA. These results indicate the possibility of delPA (163–168) and delPA (313–314) proteins being developed into nontoxic, effective and stable recombinant vaccine candidates. � 2005 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.

Of 18 vanA-positive vancomycin-susceptible Enterococcus faecium isolates, vanRS in the vanA cluster was detected in all isolates, while vanHAX was detected in only 2 isolates. Following exposure to glycopeptides, 22.2% of vancomycin-susceptible E. faecium (VSE) converted into vancomycin-resistant E. faecium. The vanA cluster of the revertant mutant was transferred to the VSE isolates.

A longitudinal analysis was carried out of the colonization by four potential respiratory pathogens - Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus - in 165 healthy children (aged 3-7 years) attending three kindergartens and 417 healthy children (aged 7-10 years) attending an elementary school in Seoul, Korea, by four consecutive examinations over 1 year. The prevalence of nasal carriers of one or more of four bacteria was found to be higher in younger children (≤7 years) (mean 68.6%) than that in older children (mean 46.8%). The mean rates of nasal carriage of Strep. pneumoniae, H. influenzae, M. catarrhalis and Staph. aureus were 16.8, 18.9, 20.2 and 18.2%, respectively. Colonization by Strep. pneumoniae, H. influenzae and M. catarrhalis was higher in pre-school children (28.6, 32.4 and 35.0%, respectively) than in school children (12.2, 13.6 and 14.3%, respectively). Carriage trends differed with age, with Strep. pneumoniae, H. influenzae and M. catarrhalis colonization decreasing with age but Staph. aureus colonization increasing. Positive associations of co-occurrence between Strep. pneumoniae, H. influenzae and M. catarrhalis were evident, with a significant negative association evident between Staph. aureus and the other three bacteria. A better understanding of the colonization and interaction of potential respiratory pathogens may be important for predicting changes in bacterial ecology and for designing control strategies that target bacterial colonization in upper respiratory tract infections.

A total of 560 Legionella species were isolated from environmental water sources from public facilities from June to September 2008 throughout South Korea. The distribution of Legionella isolates was investigated according to geographical region, facility type, and sample type. The genetic diversity of 104 isolates of Legionella pneumophila serogroup 1 (sg 1) was analyzed by sequence-based typing (SBT). L. pneumophila was distributed broadly throughout Korea, accounting for 85.0% of the isolates, and L. pneumophila sg 1 predominated in all of the public facilities except for the springs. Legionella anisa and Legionella bozemanii predominated among non- L. pneumophila species (48.1% and 21.0%, respectively). The second most dominant strain differed depending on the facility type: L. anisa was the second most dominant strain in the buildings (10.8%), L. pneumophila sg 5 in public baths (21.6%), L. pneumophila sg 6 in factories (12.0%), and L. pneumophila sg 7 in hospitals (13.1%). In the SBT analysis, 104 L. pneumophila sg 1 isolates were differentiated into 26 sequence types (STs) and categorized into 3 clonal groups (CGs) and 10 singleton STs via the eBURST V3 program. ST1, a potential founder of major CG1, was commonly distributed (48.1%). The dominant ST in hot water was ST-K1 (7, 12, 17, 3, 35, 11, 11), which was designated in this study (36.1%). The second most dominant strain differed depending on the type of facility from which the samples were obtained. The unique allelic profile of ST-K1, obtained from hot water, was not found in the European Working Group for Legionella Infections (EWGLI) SBT database.

The molecular typing of 202 Legionella pneumophila sg 1 isolates obtained from environmental water sources and clinical specimens from 1985 to 2007 was conducted using pulsed-field gel electrophoresis (PFGE). In this study, a total of 212 isolates were grouped into 35 different PFGE types and Type 1 was the predominant type, accounting for 28.7% in PFGE types. Type 1 and Type 8 were observed continuously from 1985 to 2007. In the analysis of the distribution of PFGE types in six geographic regions (Seoul-Incheon, Gangwon, Chungcheong, Gyeongsang, Jeolla, and Jeju), Type 1 was predominant throughout four regions except for Jeolla and Jeju, and Type 6 was observed in four regions except two regions (Gangwon and Jeju). Six clinical isolates belonged to PFGE Type 1, Type 6, Type 9, and Type 15. Type 1 among these types, was isolated from 3 patients with confirmed nosocomial infection at the hospital and Type 6, Type 9, and Type 15 were isolated 3 patients with suspected community-acquired infection. Type R, PFGE pattern of L. pneumophila sg 1 (ATCC 33152, Philadelphia-1), was not observed in the isolates evaluated in this study. Therefore, our results suggest that PFGE Type 1 was very prevalent in the environmental and clinical isolates in Korea. Type 1 was distributed continuously for many years throughout Korea.

Haemophilus influenzae is a frequent causative bacterial pathogen of respiratory tract infections. Resistance to beta-lactam antibiotics has been a significant clinical problem in treatment for H. influenzae respiratory infections. This study describes the serotype, antibiotic resistance and distribution of TEM-1 or ROB-1 beta-lactamase in H. influenzae isolates from local private hospitals from 2002 to 2004. Among the 100 H. influenzae respiratory isolates, only 7% were identified as serotypes a, b, e, and f, with the remaining 93% being nontypeable. Resistance to ampicillin, cefaclor, and tetracycline was 57%, 46%, and 16%, respectively. All strains were susceptible to azithromycin and ciprofloxacin, whereas amoxicillin/clavulanate, cefotaxime, and imipenem exhibited reduced susceptibilities of 99%, 99%, and 91%, respectively. All 57 ampicillin-resistant strains (minimum inhibitory concentration, MICor=4 microg/ml) were beta-lactamase-positive and possessed the TEM-1 type beta-lactamase. One beta-lactamase-positive amoxicillin/clavulanate-resistant isolate that was resistant to ampicillin (MIC128 microg/ml) had the TEM-1 type beta-lactamase and not susceptible to cefaclor and cefotaxime. Analysis of penicillin binding protein 3 revealed six residues (Asp-350, Met-377, Ala-502, Asn-526, Val-547, and Asn-569) that were substituted by Asn, Ile, Val, Lys, Ile, and Ser, respectively.

Nontuberculous mycobacteria (NTM) are a major cause of opportunistic infections in immunocompromised patients, making the reliable and rapid identification of NTM to the species level very important for the treatment of such patients. Therefore, this study evaluated the usefulness of the novel target genes tuf and tmRNA for the identification of NTM to the species level, using a PCRrestriction fragment length polymorphism analysis (PRA). A total of 44 reference strains and 17 clinical isolates of the genus Mycobacterium were used. The 741 bp or 744 bp tuf genes were amplified, restricted with two restriction enzymes (HaeIII/MboI), and sequenced. The tuf gene-PRA patterns were compared with those for the tmRNA (AvaII), hsp65 (HaeIII/HphI), rpoB (MspI/HaeIII), and 16S rRNA (HaeIII) genes. For the reference strains, the tuf gene-PRA yielded 43 HaeIII patterns, of which 35 (81.4%) showed unique patterns on the species level, whereas the tmRNA, hsp65, rpoB, and 16S rRNA-PRAs only showed 10 (23.3%), 32 (74.4%), 19 (44.2%), and 3 (7%) unique patterns after single digestion, respectively. The tuf gene-PRA produced a clear distinction between closely related NTM species, such as M. abscessus (557-84- 58) and M. chelonae (477-84-80-58), and M. kansasii (141- 136-80-63-58-54-51) and M. gastri (141-136-117-80-58-51). No difference was observed between the tuf-PRA patterns for the reference strains and clinical isolates. Thus, a diagnostic algorithm using a tuf gene-targeting PRA is a promising tool with more advantages than the previously used hsp65, rpoB, and 16S rRNA genes for the identification of NTM to the species level.

There has been a recent increase in the number of reported infections caused by nontuberculous mycobacteria (NTM), mainly in immunocompromised persons [5, 33, 17]. Thus, the reliable and rapid identification of NTM to the species level is very important, especially for patient treatment and management, owing to differences in antimicrobial susceptibility depending on the NTM species [13]. Although extensive effort has already been made to develop effective methods for identifying NTM species, perfect discrimination using one target gene is still impossible, owing to the high degree of sequence homology between closely related NTM species and newly identified mycobacteria. Therefore, reference laboratories are constantly challenged to provide accurate and rapid identifications of NTM species from clinical isolates. NTM can be categorized by the growth rate (slowly growing and rapidly growing species) and pigmentation (pigmented and nonpigmented species), which can provide helpful information for a preliminary broad classification of NTM. The conventional biochemical analysis is timeconsuming and has limited identification ability for newly described NTM [28]. As such, a commercial diagnosis kit (AccuProbe: GenProbe, Inc., San Diego, CA, U.S.A.) is also available for species identification, including specific DNA probes for a limited range of NTM (M. avium, M. intracellulare, M. kansasii, and M. gordonae), whereas a high-performance liquid chromatography (HPLC) analysis of mycolic acids is used for chemotaxonomic classification [7], which is rapid and reliable, yet still limited in its ability to distinguish and requires special instruments. Thus, several researchers have recently reported on the feasibility of identifying NTM using a molecular diagnosis method, such as a PCR-restriction fragment length polymorphism analysis (PRA) or sequencing analysis, as these approaches can identify or group several NTM relatively quickly and simply. As a result, mycobacterial housekeeping genes, such as 16S rRNA [14], 16S-23S rRNA internal transcribed spacer

Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and can cause nosocomial infections in immunocompromised patients. Recently the presence of NTM in public drinking water and hospital water distribution systems has been reported. Their ability to form biofilms and their resistance to chlorine both contribute to their survival and colonization in water distribution systems. Here we analyzed thirty-two hospital tap water samples that were collected from different locations in three hospitals so as to evaluate the prevalence of NTM species. The water samples were concentrated by membrane filtration and then eluted with sterilized water following sonication. Two-step direct PCR targeting the rpoB gene, restriction fragment length polymorphism (RFLP) using the MspI restriction enzyme, and sequence analysis were performed for identification of NTM to the species level. The sequences of each PCR product were analyzed using BLASTN. Seven samples (7/32, 21.9%) were positive for NTM as determined by nested-PCR. The PCR-RFLP results indicated five different patterns among the seven positive PCR samples. The water-born NTM were identified, including M. peregrinum, M. chelonae (2 cases), M. abscessus, M. gordonae (2 cases), and Mycobacterium sp. JLS. The direct two-step PCR-RFLP method targeting the rpoB gene was effective for the detection and the differentiation of NTM species from hospital tap water.

Six hundred fifteen isolates of Streptococcus pyogenes were collected over a 6-year period from patients with pharyngitis in Korea. All isolates were characterized in terms of their antibiotic resistance, the phenotypes of erythromycin resistance, the frequencies of erm(B), erm(A), and mef(A) genes, and the emm genotype. The prevalent emm genotypes were emm12 and emm4. Moreover, the emm12 genotype was found to be the most resistant strain to erythromycin. Among the 126 strains demonstrating resistance to erythromycin, those with erm(B) were the most prevalent, accounting for 64.3% of the total. In summary, it is suggested that the S. pyogenes pathogen isolated from pharyngitis patients in Korea developed resistant gene acquisition, as well as a resistant phenotype, according to the annual prevailing emm type. It is also suggested that the emm genotype distribution of erythromycin-resistant strains is correlated to the acquisition of resistant genes.

Pertussis is a representative vaccine-preventable disease. However, there have been recent outbreaks in countries where even higher vaccination against the disease. One reason is the emergence of antigenic variants, which are different to vaccine type. In Korea, reported cases have rapidly increased since 2009. Therefore, we analyzed genotype of strains isolated in 2011-2012 by multilocus sequence typing method. As expected, the genotype profiles of tested genes dramatically changed. The major sequence type changed from ST1 to ST2, and new sequence type (ST8) appeared. In the minimum spanning tree, recent isolates belonging to the ACC-I-ST3 subgroup were detected that were composed of ST2, ST3, and ST6. In particular, the ST2 frequency increased to 81%. The novel ST8 was linked to the increased frequency of ST2. In addition, toxic strains carrying the ptxP3 promoter type were confirmed. This ptxP3 type emerged from 2009 and its frequency had increased to 100% in 2012. Based on these results, it can be inferred that the genotypic changes in the currently circulating strains are strongly associated with the recent increasing of pertussis in Korea. Therefore, the surveillance system should be strengthened, and genetic characterization of the isolates should be expanded to the whole genome sequence level.

Background : Polymerase-chain reaction (PCR) detection is useful to diagnosis of pertussis at initial stage because the growth rate of Bordetella pertussis (B. pertussis) is relatively slow. Currently, the primer set for the insertion sequence IS481 (BP primer) is used widely for PCR detection of B. pertussis. However, the cross-reactivity of BP primer set with Bordetella holmesii (B. holmesii) was reported recently. Therefore, discrimination of B. pertussis and B. holmesii is needed in PCR step. For this reason, we developed new primer sets based on 16S rDNA sequence for diagnostic use and estimated the efficiency of these new primer sets. Materials and Methods : The specific PCR primers were designed from the aligned sequence matrix of 16S rDNA genes of various Bordetella species. The specificity of designed primers were estimated using clinically important 4 Bordetella species, B. pertussis, B. holmesii, Bordetella parapertussis (B. parapertussis) and Bordetella bronchiseptica (B. bronchiseptica). The sensitivity to B. pertussis of designed primers was also estimated and compared with BP primer set. Results : As the results, the developed new primer set successfully distinguished B. pertussis and other Bordetella species containing B. holmesii. In the sensitivity assay, the detectable limits of 16S-F2/16S-R1 primer set for B. pertussis were revealed as 5 pg of genomic DNA and 10 5