• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.575-578
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• 2000

The N-acetylneuraminate lyase(NALase) from Escherichia coli was cloned and it was compared to that from Haemophilus influenzae and Clostridium perfringens. NALase from E. coli was expressed in very high level(about 6U/mg). The ManNAc Km value of three enzymes was almost the same. Pyruvate inhibited from H. influenzae was inhibited by GlcNAc in lower level than the others. The crude extract has about 30 times more activity than the cell for the substrate and product diffusion limit problem. The pH stability of three enzymes at pH 11 was also checked for its importance in the direct synthesis of Neu5Ac from GlcNAc and pyruvate at high alkaline condition.

• Pediatric Infection and Vaccine
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• v.8 no.2
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• pp.234-240
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• 2001

Buccal cellulitis which presents with high fever and a swelling of the cheek with violaceous hue in young children is most often caused by H. influenzae. Bacteremia is common in buccal cellulitis caused by H. influenzae, and a culture of cerebrospinal fluid should be obtained because meningitis may be present despite the lack of meningeal irritation signs. Although buccal cellulitis is considered to be one of the important manifestations of H. influenzae infection, only two cases have been reported in Korea yet. We experienced a case of buccal cellulitis with H. influenzae bacteremia in an immunocompetent girl of 18-month-old. She was presented with high fever followed by rapidly progressive swelling and tenderness of both cheeks with violaceous hue in four hours. The blood culture revealed H. influenzae type b. There was no concurrent otitis media, sinusitis, or meningitis and no portal of entry was identified. Fever subsided two days after starting intravenous cefotaxime. Intravenous cefotaxime was subsequently changed to oral cefixime, and antibiotics were administered for a total of two weeks. We report this case with a review of related literature.

• Pediatric Infection and Vaccine
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• v.27 no.3
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• pp.147-157
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• 2020

Purpose: We investigated the trend of antibiotic susceptibility of Haemophilus influenzae over 5 consecutive years. Methods: We analyzed the antibiotic susceptibility of H. influenzae isolated from children aged <18 years, who were admitted to the Asan Medical Center Children's Hospital from March 2014 to April 2019. Antibiotic susceptibility of H. influenzae was determined by the disk diffusion test according to the European Committee on Antimicrobial Susceptibility Testing guidelines. Results: Excluding duplicates, 69 isolates were obtained over the past 5 years. The median age of the patients was 5 years (range, 2.8-8.6 years). The antibiotic susceptibility patterns were as follows: ampicillin (AMP)-susceptible/amoxicillin-clavulanate (AMC)-susceptible (AS/ACS; n=15 [21.7%]), AMP-resistant/AMC-susceptible (AR/ACS; n=21 [30.4%]), and AMP-resistant/AMC-resistant (AR/ACR; n=33 [47.8%]). The prevalence of isolates with AR/ACR phenotype tended to increase from 42.1% in 2014-2015 to 54.5% in 2018-2019 (P=0.342). Compared to 2014-2015, the resistance rates to cefuroxime and ceftriaxone in 2018-2019 increased from 31.6% to 77.3% and from 0.0% to 59.1%, respectively (P=0.003 and P<0.001, respectively). Conclusions: Over the last 5 years, H. influenzae isolates with AR/ACR phenotype and ceftriaxone resistance were frequently observed at our institute. The incidence of resistance to cefuroxime and ceftriaxone has increased significantly.

• Journal of Microbiology
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• v.41 no.2
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• pp.78-82
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• 2003

We identified two proteins in Haemophilus influenzae Rd that exhibited high similarity to two major serine transporters of Escherichia coli (SstT and SdaC). Then, we investigated serine transport in H. influenzae Rd and detected $Na^{+}$-stimulated L-serine transport activity. The optimum NaCl concentration for this stimulation was about 20 mM. The uptake of $Na^{+}$ by H. influenzae Rd was found to be elicited by L-serine influx, which supports the idea that L-serine is transported by a mechanism of $Na^{+}$/serine symport. No uptake of $H^{+}$ elicited by L-serine influx was detected. $Na^{+}$/serine symport activity was not inhibited by other amino acids such as L-threonine or D-serine. Two distinct Km values were obtained from the kinetic analysis of serine transport. Thus, two serine transport pathways may exist in H. influenzae Rd, and it appears that both systems are stimulated by $Na^{+}$.

• Journal of Microbiology
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• v.41 no.3
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• pp.202-206
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• 2003

A protein, exhibiting a high similarity to the major serine transporter of Escherichia coli, SstT, was found in Haemophilus influenzae Rd. A Na$\^$＋/-stimulated serine transport activity was also detected in the cells. The gene (sstT) for the Na$\^$＋//serine symporter from the chromosome of H. influenzae was cloned, and the properties of the transporter investigated. The serine transport activity was stimulated by Na$\^$＋/. The uptake of Na$\^$＋/ was elicited by the addition of serine or threonine into the cells, supporting the idea that these amino acids are transported by a mechanism of Na$\^$＋//substrate symport. No uptake of H$\^$＋/ was elicited by the influx of serine. The serine transport via the SstT of H. influenzae was inhibited by excess threonine, which was used as another substrate. The $K_{m}$ and the $V_{max}$ values for the serine transport were 2.5 ${\mu}$M and 14 nmol/min/mg protein, respectively.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.520-524
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• 2004

A protein that exhibited a high similarity to a major serine transporter of Escherichia coli, SdaC, was found in Haemophilus injluenzae Rd. Also, $Na^+$-stimulated serine transport activity was detected in the cells. The sdaC of H. injluenzae was cloned and the properties of the transporter were investigated. The activity of serine transport was stimulated by $Na^+$. Uptake of $Na^+$ elicited by L-serine influx into cells was also observed, which supports the idea that L-serine is transported by a mechanism of $Na^+$serine symport. No uptake of $H^+$ elicited by L-serine influx was detected. This result was not consistent with that obtained with the homologous protein, SdaC of E. coli, which uses $H^+$as a coupling cation. The serine transport via the SdaC of H. influenzae was not inhibited by other amino acids such as threonine or D-serine like the SdaC of E. coli. Thus, the SdaC of H. influenzae is a $Na^+$-dependent L-serine specific symporter and an unusual natural mutant. The $K_m$ and the $V_{max}$, value for the serine transport in the SdaC of H. influenzae were $7.6\mu$M and 22.9 nmol/min/mg protein, respectively.

• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.103-110
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• 2003

The regulation of pyrimidine nucleotide synthesis has been proved to be controlled by a regulatory protein PyrR-mediated attenuation in the Gram-positive bacteria. After several bacterial genome sequencing projects, we have discovered the PyrR orthologues in the databases for Haemophilus influenzae and Synechocystis and sp. PCC6803 genome sequences. To investigate whether these PyrR orthologue proteins regulate pyrimidine nucleotide synthesis as well as the cases of Bacillus, the PyrR regions of each strains were amplified by PCR and cloned with pUC19 or T-vector in Escherichia coli and with a shuttle vector pHPS9 for E. coli and B. subtilis. For the regulation test of the PyrR orthologues, the aspartate-transcarbamylase (ATCase) assay was carried out. From the results of the ATCase assay, it was confirmed that Synechocystis sp. PCC6803 could not restore by pyrimidines to a B. subtilis, PyrR but H. influenzae PyrR could. For Purification of PyrR orthologue proteins, PyrR orthologue genes were cloned into the expression vector (pET14b). Over-expressed product of PyrR orthologue genes was purified and analyzed by the SDS-PACE. The purified PyrR orthologue proteins from H. influenzae and Synechocystis sp. PCC6803 turned out to be molecular mass of 18 kDa and 21 kDa, respectively. The result of uracil phosphoribosyl transferase (UPRTase) assay with purified PyrR orthologue proteins showed that H. influenzae PyrR protein only has UPRTase activity. In addition, we could predict several regulatory mechanisms that PyrR orthologue proteins regulate pyrimidine de novo synthesis in bacteria, through phylogenetic analysis for PyrR orthologue protein sequences.

• Korean Journal of Microbiology
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• v.43 no.1
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• pp.19-22
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• 2007

Acetohydroxyacid synthase (E.C.2.2.1.6., AHAS) is the enzyme that catalyses the first step in the synthesis of the branched-chain amino acids valine, leucine and isoleucine. The AHAS gene (TIGR access code HI2585) from Heamophilus influenzae was cloned into the bacterial expression vector pET-28a and expressed in the Escherichia coli strain BL21(DE3). The expressed enzyme was purified by $Ni^{2+}-charged$ HiTrap chelating HP column. The purified enzyme appears as a single band on SDS-PAGE with a molecular mass of about 63.9 kDa. The enzyme exhibits absolute dependence on the three cofactors FAD, $MgCl_{2}$ and thiamine diphosphate for activity. Specific activity of purified enzyme has 3.22 unit/mg and optimum activity in the pH 7.5 at $37^{\circ}C$. This enzyme activity has an effect on the buffer. When comparing the enzyme activity against the organic solvent, it followed in type and the difference it is but even from the aqueous solution where the organic solvent is included with the fact that the enzyme activity is maintained.

• Clinical and Experimental Pediatrics
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• v.52 no.12
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• pp.1358-1363
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• 2009

Purpose:The aim of this study was to determine the prevalence of asymptomatic nasopharyngeal carriages in children using a multiplex reverse transcriptase-polymerase chain reaction (mRT-PCR) assay kit. Methods:We obtained nasopharyngeal swabs from 33 children without any underlying disease from July 25 to July 28, 2008. The children were free from the signs of respiratory tract infections at the time of sampling. DNA was extracted from the swabs and subjected to multiplex RT-PCR using a primer set for the detection of pneumococci ($Seeplex^{(R)}$ PneumoBacter ACE Detection Seegene, Seoul, Korea). The amplified PCR products were separated on 2% agarose gels and stained with either ethidium bromide or screen tape system (Lab901 Scotland, UK). Results:A total of 33 children (male, 15 female, 18) aged between 3.2 and 16.3 (median, 8.2) years were included in this study. The mRT-PCR detected colonized bacteria (Streptococcus pneumoniae, Hemophilus influenzae, Chlamydia pneumoniae, and Bordetella pertussis) in 30 children (90.9%). Of these, 13 children (39.4%) showed more than 2 bacteria: 12 children were positive for 2 bacteria (S. pneumoniae and H. influenzae) and 1 child was positive for 3 bacteria (S. pneumoniae, H. influenzae, and C. pneumoniae). Conclusion:mRT-PCR was found to be a sensitive tool for the detection of asymptomatic nasopharyngeal carriages. Clinical significances of the bacteria detected by mRT-PCR will have to be evaluated in the future.

• Clinical and Experimental Pediatrics
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• v.46 no.11
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• pp.1085-1088
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• 2003

Purpose : This study was performed to assess the difference of organisms causing bacterial meningitis according to time. Methods : We analyzed retrospectively 40 medical records of bacteriologically proven meningitis from 1992 to 2002. We divided them into two groups; neonate's group(14 cases), and children's group(26 cases). The results of the neonate's group were compared with those of previously reported articles in Korea, in 1970s-1980s. The causative agents of the children's group were analyzed according to the stage before and after the introduction of H. influenza type b(Hib) vaccine. Results : In neonates, Group B streptococci(GBS) was the most common cause of bacterial meningitis. There was a trend in Korea that major causative agents of neonatal bacterial meningitis have changed from gram negative bacteria including E. coli to gram positive bacteria including GBS. In children, H. influenzae was isolated in six out of 11 cases(55%) in 1992-95, before the introduction of Hib vaccine, while two out of seven(29%) were isolated in 1999-2002, after the introduction of the Hib vaccine. Conclusion : Our study showed that the most common agent of neonatal bacterial meningitis was GBS. There was a trend that after the introduction of Hib vaccine, the incidence of H. influenza meningitis decreased in children.