A Novel Time-Resolved Fluorescence Resonance Energy Transfer Assay for the Discovery of Small-Molecule Inhibitors of HIV-1 Tat-Regulated Transcription.

Human immunodeficiency virus-1 (HIV-1) transactivator (Tat)-mediated transcription is essential for HIV-1 replication. It is determined by the interaction between Tat and transactivation response (TAR) RNA, a highly conserved process representing a prominent therapeutic target against HIV-1 replication. However, owing to the limitations of current high-throughput screening (HTS) assays, no drug that disrupts the Tat-TAR RNA interaction has been uncovered yet. We designed a homogenous (mix-and-read) time-resolved fluorescence resonance energy transfer (TR-FRET) assay using europium cryptate as a fluorescence donor. It was optimized by evaluating different probing systems for Tat-derived peptides or TAR RNA. The specificity of the optimal assay was validated by mutants of the Tat-derived peptides and TAR RNA fragment, individually and by competitive inhibition with known TAR RNA-binding peptides. The assay generated a constant Tat-TAR RNA interaction signal, discriminating the compounds that disrupted the interaction. Combined with a functional assay, the TR-FRET assay identified two small molecules (460-G06 and 463-H08) capable of inhibiting Tat activity and HIV-1 infection from a large-scale compound library. The simplicity, ease of operation, and rapidity of our assay render it suitable for HTS to identify Tat-TAR RNA interaction inhibitors. The identified compounds may also act as potent molecular scaffolds for developing a new HIV-1 drug class.

Characteristics of Escherichia coli Urine Isolates and Risk Factors for Secondary Bloodstream Infections in Patients with Urinary Tract Infections.

Escherichia coli is responsible for more than 80% of all incidences of urinary tract infections (UTIs). We assessed a total of 636 cases of patients with E. coli UTIs occurring in June 2019 in eight tertiary hospitals in South Korea for the traits of patients with E. coli UTIs, UTI-causative E. coli isolates, and risk factors associated with bloodstream infections (BSIs) secondary to UTIs. Antimicrobial susceptibility testing was conducted using the disc diffusion method, and the genes for extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated ampC genes were screened by using PCR and sequencing. Multilocus sequence typing and virulence pheno-/genotyping were carried out. A total of 49 cases developed BSIs. The E. coli urine isolates primarily comprised sequence type 131 (ST131) (30.0%), followed by ST1193, ST95, ST73, and ST69. Three-quarters of the ST131 H30Rx isolates possessed the blaCTX-M-15-like gene, whereas 66% of H30R and 50% of H41 isolates possessed the blaCTX-M-14-like gene. All the ST1193 isolates showed biofilm formation ability, and three-quarters of the ST73 isolates exhibited hemolytic activity with high proportions of papC, focG, and cnf1 positivity. The prevalence of the ST131 H41 sublineage and its abundant CTX-M possession among the E. coli urine isolates were noteworthy; however, no specific STs were associated with bloodstream invasion. For BSIs secondary to UTIs, the papC gene was likely identified as a UTI-causative E. coli-related risk factor and urogenital cancer (odds ratio [OR], 12.328), indwelling catheter (OR, 3.218), and costovertebral angle tenderness (OR, 2.779) were patient-related risk factors. IMPORTANCE Approximately half of the BSIs caused by E. coli are secondary to E. coli UTIs. Since the uropathogenic E. coli causing most of the UTIs is genetically diverse, understanding the risk factors in the E. coli urine isolates causing the BSI is important for pathophysiology. Although the UTIs are some of the most common bacterial infectious diseases, and the BSIs secondary to the UTIs are commonly caused by E. coli, the assessments to find the risk factors are mostly focused on the condition of patients, not on the bacterial pathogens. Molecular epidemiology of the UTI-causative E. coli pathogens, together with the characterization of the E. coli urine isolates associated with the BSI secondary to UTI, was carried out, suggesting treatment options for the prevalent antimicrobial-resistant organisms.

Phylogenetic Comparison and Characterization of an mcr-1-Harboring Complete Plasmid Genome Isolated from Enterobacteriaceae.

Global dissemination of mobilized colistin resistance (mcr)-1-carrying plasmids has been reported. This study aimed to investigate the global dissemination of these plasmids using whole genome sequencing to provide better understanding on genetic characteristics. Sixty-seven complete plasmid genomes harboring mcr-1 were obtained. Phylogeny was built against full plasmid genomes. Different replicon types of plasmid were compared in terms of antimicrobial resistance genes (ARGs), insertion sequence, and other functional genes. Five different replicon types of plasmid (IncX4, IncI2, IncP1, IncHIA, and IncFIB) were found to harbor mcr-1. IncX4 and IncI2 types of plasmid were well clustered in accordance with the country where they were isolated (and not as IncHIA and IncFIB). Three insertion sequences (ISApl1, ISKpn26, and IS1294) were identified in up- and/or downstream of mcr-1. Plasmids IncX4 and IncI2 were observed across the sample origin. Plasmids IncX4 showed high uniformity regardless of the origin of isolates and harbored H-NS coding genes, a facilitator for successful plasmid transfer. All three insertion sequences were observed in IncI2 plasmids. IncHI2 plasmids harbored various ARGs in addition to mcr-1. Our results elucidate the characteristics and phylogenetic relationships of complete mcr-1-harboring plasmids, indicating that global dissemination of mcr-1 is primarily owing to plasmid transfer rather than clonal spread.

The Global Landscape of Pediatric Bacterial Meningitis Data Reported to the World Health Organization-Coordinated Invasive Bacterial Vaccine-Preventable Disease Surveillance Network, 2014-2019.

BACKGROUND: The World Health Organization (WHO) coordinates the Global Invasive Bacterial Vaccine-Preventable Diseases (IB-VPD) Surveillance Network to support vaccine introduction decisions and use. The network was established to strengthen surveillance and laboratory confirmation of meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis. METHODS: Sentinel hospitals report cases of children <5 years of age hospitalized for suspected meningitis. Laboratories report confirmatory testing results and strain characterization tested by polymerase chain reaction. In 2019, the network included 123 laboratories that follow validated, standardized testing and reporting strategies. RESULTS: From 2014 through 2019, >137 000 suspected meningitis cases were reported by 58 participating countries, with 44.6% (n = 61 386) reported from countries in the WHO African Region. More than half (56.6%, n = 77 873) were among children <1 year of age, and 4.0% (n = 4010) died among those with reported disease outcome. Among suspected meningitis cases, 8.6% (n = 11 798) were classified as probable bacterial meningitis. One of 3 bacterial pathogens was identified in 30.3% (n = 3576) of these cases, namely S. pneumoniae (n = 2177 [60.9%]), H. influenzae (n = 633 [17.7%]), and N. meningitidis (n = 766 [21.4%]). Among confirmed bacterial meningitis cases with outcome reported, 11.0% died; case fatality ratio varied by pathogen (S. pneumoniae, 12.2%; H. influenzae, 6.1%; N. meningitidis, 11.0%). Among the 277 children who died with confirmed bacterial meningitis, 189 (68.2%) had confirmed S. pneumoniae. The proportion of pneumococcal cases with pneumococcal conjugate vaccine (PCV) serotypes decreased as the number of countries implementing PCV increased, from 77.8% (n = 273) to 47.5% (n = 248). Of 397 H. influenzae specimens serotyped, 49.1% (n = 195) were type b. Predominant N. meningitidis serogroups varied by region. CONCLUSIONS: This multitier, global surveillance network has supported countries in detecting and serotyping the 3 principal invasive bacterial pathogens that cause pediatric meningitis. Streptococcus pneumoniae was the most common bacterial pathogen detected globally despite the growing number of countries that have nationally introduced PCV. The large proportions of deaths due to S. pneumoniae reflect the high proportion of meningitis cases caused by this pathogen. This global network demonstrated a strong correlation between PCV introduction status and reduction in the proportion of pneumococcal meningitis infections caused by vaccine serotypes. Maintaining case-based, active surveillance with laboratory confirmation for prioritized vaccine-preventable diseases remains a critical component of the global agenda in public health.The World Health Organization (WHO)-coordinated Invasive Bacterial Vaccine-Preventable Disease (IB-VPD) Surveillance Network reported data from 2014 to 2019, contributing to the estimates of the disease burden and serotypes of pediatric meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis.

Cluster of serogroup W-135 meningococcal disease in 3 military recruits.

We describe a group of 3 cases of invasive meningococcal disease that occurred in a military training camp in April 2011. All three patients were hospitalized. Ultimately, two patients recovered and one died. One patient had meningitis, one patient had septicemia and meningitis, and the other had no definite septicemia or meningitis. Neisseria meningitidis serogroup W-135 was detected in the serum and cerebrospinal fluid (CSF) of all patients by real-time polymerase chain reaction. In the one case of mortality, two strains were isolated from the patient's blood and CSF. Using multilocus sequence typing analysis, these strains were identified as a novel sequence type, ST-8912. Special attention is required for the meningococcal disease in military camp because the military personnels are in high risk of contact transmission.

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.

High prevalence of multiresistance in levofloxacin-nonsusceptible Streptococcus pneumoniae isolates in Korea.

Korea exhibits the highest rates of multidrug resistance among Streptococcus pneumoniae. The increasing use of levofloxacin has raised concern about the dissemination of levofloxacin resistance in dominant multidrug-resistant (MDR) clones of our pneumococcal population. A total of 50 levofloxacin-nonsusceptible S. pneumoniae (MIC, ≥4 µg/mL) collected from a multihospital network from 1996 to 2006 were analyzed for serotype, antibiotic resistance profile, quinolone resistance-determining region mutation, and multilocus sequence type. Most levofloxacin-nonsusceptible S. pneumoniae (94.0%) exhibited an MDR phenotype. This phenotype was closely associated with a limited number of epidemic MDR clones that are well-known key agents of the global spread of antimicrobial resistance in S. pneumoniae. However, the clonal dissemination of levofloxacin-nonsusceptible S. pneumoniae was rare. Levofloxacin-nonsusceptible clones with nonvaccine serotypes increased during the post-vaccine era in this study. This result suggests that Korean clinicians must be aware of the levofloxacin resistance trend and need to be more prudent for the first choice of fluoroquinolone for empiric treatment of respiratory tract infections in clinical setting. Moreover, the emergence of new clones and their variations may be more frequently associated with resistance under this selective pressure, such as the introduction of a 7-valent pneumococcal conjugate vaccine into our community.

Prevalent Multidrug-resistant Nonvaccine Serotypes in Pneumococcal Carriage of Healthy Korean Children Associated with the Low Coverage of the Seven-valent Pneumococcal Conjugate Vaccine.

OBJECTIVES: Our previous longitudinal multicenter-based carriage study showed that the average carriage rate of Streptococcus pneumoniae was 16.8% in 582 healthy children attending kindergarten or elementary school in Seoul, Korea. We assessed serotype-specific prevalence and antimicrobial resistance among colonizing pneumococcal isolates from young children in the era of low use of the seven-valent pneumococcal conjugate vaccine (PCV7). METHODS: Serotypes were determined by an agglutination test with specific antisera or by a multiplex polymerase chain reaction (PCR) assay. An antimicrobial susceptibility test was performed with broth microdilution in Korean 96-well panels from Dade-MicroScan (Sacramento, CA, USA). RESULTS: Pneumococcal colonization patterns were dynamic and longterm persistent carriage was rare, which indicated a sequential turnover of pneumococcal strains. Of the 369 pneumococci (except for 23 killed isolates), 129 (34.9%) isolates were PCV7 vaccine serotypes (VTs); 213 (57.8%) isolates were nonvaccine serotypes (NVTs); and the remaining 27 (7.2%) isolates were nontypable (NT). The highest rates of multidrug resistance (MDR) were observed in VTs (86.0%; 111/129 isolates) and NVTs (70.0%; 149/213 isolates). CONCLUSION: This study overall showed the frequent carriage of VTs and NVTs with MDR in healthy children attending kindergarten or elementary school. Efforts should be directed toward reducing the extensive prescription of antibiotics and using new broader vaccines to reduce the expansion of MDR strains of NVTs in our community.

Carriage rates and serogroups of Neisseria meningitidis among freshmen in a University dormitory in Korea.

PURPOSE: Neisseria meningitidis is a leading cause of bacterial meningitis in young adults. University students, especially those living in dormitories, have been known to be at increased risk of meningococcal disease. We performed a longitudinal study to determine the carriage rates of N. meningitidis and the changes thereof. MATERIALS AND METHODS: We recruited Inha University freshmen who were, at that time, admitted to a student dormitory. A pharyngeal swab was taken from all participant who were also asked to complete a questionnaire. This was repeated four weeks later. RESULTS: A total of 136 students were enrolled at the first culture. After four weeks, 128 students were enrolled, including 106 re-participants. The overall carriage rates changed from 11.8% to 14.1%. In analysis of the 106 re-participants, "visiting to pubs" was associated with carriage of N. meningitis for both the first (p=0.047) and second cultures (p=0.026). Serogroup C was found to be the most frequent serogroup (5 isolates), while 3 isolates were found from serogroup B. The most prevalent PorA types were P1.22,14-6 (4 isolates) and P1.19,15 (3 isolates). The DNA sequences of PorA VR2 were changed in 2 students during prolonged carriage. CONCLUSION: The meningococcal carriage rate among first year university students who resided in a dormitory did not significantly increase over 4-week interval between cultures, which is markedly different from those reported in Western studies. Close social contact appeared to be related with carriage. Our data also revealed diversity in PorA types, suggesting the possibility of rapid mutation of the PorA gene during the 4-week interval.

Nasal colonization by four potential respiratory bacteria in healthy children attending kindergarten or elementary school in Seoul, Korea.

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.

Streptococcus pneumoniae pep27 mutant as a live vaccine for serotype-independent protection in mice.

Streptococcus pneumoniae (pneumococcus) is responsible for significant morbidity and mortality in worldwide. After introduction of current pneumococcal vaccines, a marked decrease in the incidence of pneumococcal disease was observed. Unfortunately, serotype shifts in carriage and disease, including capsular switch and presence of antimicrobial resistance, have been found. Here we report live attenuated vaccine strain which is avirulent and can protect from systemic and mucosal pneumococcal diseases. Pep27, an autolysis-inducing factor of S. pneumoniae is known to mediate LytA-dependent and -independent lysis and it was thus expected to effect virulence. The loss of Pep27 had a much larger than expected decrease in virulence and has made the Pep27 mutant strain sufficiently avirulent to be used as a live vaccine. The pep27 mutation unexpectedly had lower level of capsular polysaccharide than the wild type (type 2, D39) strain. Moreover, the pep27 mutant showed rapid clearance by 24 h post intranasal infection, and was not detected in lung and blood suggesting that mutant could not invade into the tissue. Even when 2×10(8)CFU were injected intravenously the mutant was not detected in the blood or brain after 4 h. Whereas 4 h after injection of 6×10(6) CFU of the wild type parent D39 strain, bacteremia was readily detected. Two dose intranasal immunizations with the live pep27 mutant in the absence of adjuvant elicited IgG antibody and serotype-independent protection against lethal intranasal challenge. Thus Pep27 was essential for virulence, and intranasal immunization with the pep27 mutant could provide protective immunity.

Prevalence of serotype and multidrug-resistance of Streptococcus pneumoniae respiratory tract isolates in 265 adults and 36 children in Korea, 2002-2005.

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 concentration >or=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.

Serotype distribution and beta-lactam resistance in Haemophilus influenzae isolated from patients with respiratory infections in 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, MIC>or=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 (MIC>128 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.

Antimicrobial resistance in Haemophilus influenzae respiratory tract isolates in Korea: results of a nationwide acute respiratory infections surveillance.

Antimicrobial susceptibility patterns and beta-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. beta-Lactamase production (52.4%) was the main mechanism of ampicillin resistance, affecting 96.1% of TEM-1-type beta-lactamase. According to their beta-lactam resistance mechanisms, all isolates were classified into the following groups: beta-lactamase-negative, ampicillin-sensitive (BLNAS) strains (n = 224; 41.5%); beta-lactamase-positive, ampicillin-resistant (BLPAR) strains (n = 255; 47.2%); beta-lactamase-negative, ampicillin-resistant (BLNAR) strains (n = 33; 6.1%); and beta-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 mug/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.

Distribution of capsular serotypes and macrolide resistance mechanisms among macrolide-resistant Streptococcus pneumoniae isolates in Korea.

The mechanism of macrolide resistance was investigated in 251 Streptococcus pneumoniae isolates with reduced susceptibility to erythromycin (erythromycin-resistant S. pneumoniae [ERSP]) collected during the period from 2000 to 2004 in Korea. Among these strains, erm(B) was the most prevalent pneumococcal macrolide resistance genotype. In particular, dual mechanisms of both the erm(B) and mef(A) genes were detected in 77 (30.7%) of 251 ERSP isolates. All of the 77 ERSP isolates, with dual erm(B) and mef(A), showed resistance to 2 or more antimicrobial agents, including penicillin, cefotaxime, clindamycin, tetracycline, and levofloxacin. Serotypes 19F, 23F, 19A, 14, 11A, 6B, 6A, and 9V accounted for 73.3% of ERSP isolates. Most of the strains with serotypes 19F (77.2%) or 19A (87.5%) had the dual erm(B) and mef(A) genes. The prevalence and spread of serotype 19F or 19A isolates may have contributed to the high rate of macrolide-resistant pneumococci in Korea. In addition, we identified the emergence of a macrolide-nonsusceptible nonvaccine serotype 35B, which carries mef(A)-mediated resistance to macrolides. These findings emphasize the need for a continuous monitoring of macrolide-resistant S. pneumoniae in Korea.

Serological and genetic characterization of meningococcal isolates in Korea.

Meningococcal disease has been regarded as a very rare infection in Korea. Until now, there have been no reports on the serological or genetic characterization of Neisseria meningitidis isolates in Korea. This study was the first report of the serogroup, PorA VR subtype, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and antimicrobial susceptibility of N. meningitidis isolates collected from 2002 to 2003. Of 11 meningococcal isolates, serogroup Y was found to be the most frequent (nine isolates). In addition, one isolate was from serogroup B and one was from serogroup 29E. Four isolates showed a reduced sensitivity to penicillin G. However, all strains tested were susceptible to chloramphenicol, cefotaxime, ciprofloxacin, and rifampin. Among the 11 isolates, seven PorA types were identified. P1.5-1, 2-2 was the most prevalent PorA type, accounting for 55.6% of the serogroup Y isolates. In terms of PFGE patterns, nine isolates of serogroup Y were divided into three clusters, but the isolates shared a high level of PFGE pattern similarity. The serogroup Y isolates were characterized as ST-1625 (five strains) and ST-23 (four). They belonged to the ST-23 complex/Cluster A3. In this study, the ST-23 complex/Cluster A3 was prevalent, with the PorA type P1.5-1, 2-2 accounting for 55.6% of the nine serogroup Y strains. Also, we identified the hypervirulent lineage strain such as ST-6667 of ST-41/44 complex/Lineage 3 in Korea. The results of this study show the need for comprehensive epidemiological surveillance to monitor any changes in the meningococcal disease situation so that prompt intervention can be initiated.

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.

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.

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.

Proteomic analysis of growth phase-dependent proteins of Streptococcus pneumoniae.

Streptococcus pneumoniae is an important human pathogen that causes a variety of diseases, such as pneumonia, bacteremia, meningitis, otitis media, and sinusitis, in both adults and children. The global pattern of growth phase-dependent protein expression of S. pneumoniae during in vitro culture was analyzed using 2-DE combined with MALDI-TOF MS and LC/ESI-MS/MS. Several protein production patterns were observed at four time points throughout the growth stage, although some protein levels did not change significantly. We focused on the switch in protein expression at the transition from log growth phase to stationary phase. Proteins that were significantly induced or repressed at this point are likely to be involved in central intermediary metabolism, amino acid synthesis, nucleotide, and fatty acid metabolism, cell wall synthesis, protein degradation, and stress responses. This global expression profiling approach has revealed previously unrecognized relationships between proteins in the life of this pathogen.