폐수처리시스템에서의 그람 양성 세균 모니터링 방법

남지현,배우근,이동훈,Nam, Ji-Hyun,Bae, Woo-Keun,Lee, Dong-Hun 한국미생물학회 2011 미생물학회지 Vol.47 No.1

폐수 처리 시스템의 그람 양성 세균을 간편하고 신속하게 모니터링할 수 있는 그람 염색 방법을 개발하였다. 각각 4종류의 그람 양성 세균과 음성 세균의 배양액을 polyethersulfone membrane filter와 Toluidine Blue-O로 여과 및 염색하였다. 염색된 여과막을 건조시키고 스캔하였으며, 영상 분석 프로그램을 이용하여 빨강색, 녹색, 파랑색의 강도를 측정하는 정량적 색상 이미지 분석법을 개발하였다. 그람 양성 세균의 경우 그람 음성 세균보다 빨간색과 초록색 수치가 높게 나타났다. 본 방법을 그람 양성 세균이 혼합된 활성슬러지에 적용한 결과, 활성슬러지의 경우 플록의 불규칙한 크기와 형태로 측정이 어렵다 할지라도 플록 분산 기술을 이용하여 활성슬러지의 그람 양성 세균의 변화를 모니터링 할 수 있었다. 활성슬러지에서도 그람 양성 세균의 비율이 높아질수록 빨간색과 초록색 수치가 증가하였다. 본 연구에서 개발된 방법은 폐수처리 시스템에 존재하는 그람 양성 세균을 신속하고 정량적으로 모니터링 할 수 있다. A simple and rapid method was developed for monitoring of Gram-positive bacteria in the wastewater treatment system. Culture suspensions of 4 Gram-positive and 4 Gram-negative strains were filtrated and stained with a polyethersulfone membrane filter and Toluidine Blue-O. To establish quantitative color image analysis, the intensity value of RGB (red-green-blue) color of a scanned filter image was analyzed with a photographic program. Red and green color values of Gram-positive bacteria were higher than those of Gram-negative bacteria. This method was applied to the activated sludge mixed with the Gram-positive bacteria. Although evaluation was difficult due to the irregular size and shape of flocs, the population of Gram-positive bacteria in the activated sludge could be monitored with floc dispersion technique. The more amounts of Gram-positive bacteria in the activated sludge led to the increase of red and green color values. This method provides a rapid and quantitative measurement of Gram-positive bacteria within the wastewater treatment systems.

Gram-positive bacteria produce membrane vesicles: Proteomics-based characterization of Staphylococcus aureus-derived membrane vesicles

Lee, Eun-Young,Choi, Do-Young,Kim, Dae-Kyum,Kim, Jung-Wook,Park, Jung Ok,Kim, Sungjee,Kim, Sang-Hyun,Desiderio, Dominic M.,Kim, Yoon-Keun,Kim, Kwang-Pyo,Gho, Yong Song WILEY-VCH Verlag 2009 Proteomics Vol.9 No.24

<P>Although archaea, Gram-negative bacteria, and mammalian cells constitutively secrete membrane vesicles (MVs) as a mechanism for cell-free intercellular communication, this cellular process has been overlooked in Gram-positive bacteria. Here, we found for the first time that Gram-positive bacteria naturally produce MVs into the extracellular milieu. Further characterizations showed that the density and size of Staphylococcus aureus-derived MVs are both similar to those of Gram-negative bacteria. With a proteomics approach, we identified with high confidence a total of 90 protein components of S. aureus-derived MVs. In the group of identified proteins, the highly enriched extracellular proteins suggested that a specific sorting mechanism for vesicular proteins exists. We also identified proteins that facilitate the transfer of proteins to other bacteria, as well to eliminate competing organisms, antibiotic resistance, pathological functions in systemic infections, and MV biogenesis. Taken together, these observations suggest that the secretion of MVs is an evolutionally conserved, universal process that occurs from simple organisms to complex multicellular organisms. This information will help us not only to elucidate the biogenesis and functions of MVs, but also to develop therapeutic tools for vaccines, diagnosis, and antibiotics effective against pathogenic strains of Gram-positive bacteria.</P>

Bacteremia in Pediatric Cancer Patients : Causative Organisms and Antibiotic Sensitivities

김용한,이현동,하정옥,Kim, Yong-Han,Lee, Hyun-Dong,Hah, Jeong-Ok The Korean Pediatric Society 2005 Clinical and Experimental Pediatrics (CEP) Vol.48 No.6

목 적 : 면역 저하된 소아 암 환자에서 균혈증은 조기에 적절한 치료를 하지 못하면 이환율과 사망률이 높아 질 수 있는 질환으로서 호발 원인균의 발생 빈도 및 항생제에 대한 감수성은 지역적 시기적으로 변화하는 경향이 있으므로 이를 조사하여 경험적 항생제 선택에 도움을 주고자 하였다. 방 법 : 1995년 9월부터 2003년 8월까지 영남대학교 의과대학부속병원 소아과에 입원한 소아 암 환자를 대상으로 병력지와 혈액배양검사 결과를 조사하여 균혈증이 확진된 39명, 58례를 대상으로 원인 균의 종류 및 항생제 감수성을 조사 분석하였다. 결 과 : 소아 암 환자에서 균혈증의 발생빈도는 균혈증이 의심되어 혈액배양을 시행한 1,022례 중 58례로 5.7%였다. 균혈증을 일으킨 가장 흔한 원인 균은 그람양성균으로 총 37례(63.8%) 였고 그람음성균은 총 21례(36.2%)였다. 그람양성균의 경우 Staphylococcus epidermidis (22례, 37.9%), Staphylococcus aureus (10례, 17.3%), Streptococcus (5례, 8.6%) 순이었고, 그람음성균의 경우 Escherichia coli (7례, 12%), Enterobacter (4례, 6.9%), Klebsiella (4례, 6.9%), Serratia (2례, 3.5%), Acinetobacter (2례, 3.5%), Proteus (1례, 1.7%), Morganella morganii (1례, 1.7%) 순으로 나타났다. 항생제 감수성 검사에서는 그람양성균 37례 중 6례(16%)에서만 penicillin 감수성을 나타내었고, 15례(40%)에서 oxacillin 감수성을 나타내었다. Staphylococcus aureus는 1례를 제외한 모든 예에서 vancomycin에, Staphylococcus epidermidis는 1례를 제외한 모든 예에서 teicoplanin에 감수성을 나타내었다. 그람음성균의 경우, 21례 중 2례(10%)만이 cefotaxime에, 4례(19%)에서만 ceftazidime에 감수성을 나타내었다. Aminoglycoside에는 21례 중 6례(29%) 에서만 감수성을 나타내었으며, 21례 모두에서 imipenem에 감수성을, 2000년 이후부터 감수성 검사를 시행한 meropenem에도 모든 예에서 감수성을 나타내었다. 결 론: 균혈증이 의심되는 소아 암 환자에서 그람양성균에 의한 균혈증이 증가하는 점과 그람양성균과 그람음성균 모두 항생제 다제내성 빈도가 증가하고 있음을 유념하여 적절한 항생제를 선택해야 할 것이다. Purpose : Bacteremia in immunocompromised pediatric cancer patients can lead to high morbidity and mortality, if not treated early and properly. The incidence and antibiotic sensitivities to common pathogens of bacteremia in pediatric cancer patients are liable to change, according to region and time. We investigated the causative organisms and antibiotic sensitivities of bacteremia in pediatric cancer patients to assess the adequacy of empiric antimicrobial therapy. Methods : From September 1995 to August 2003, we retrospectively evaluated 58 episodes in 39 pediatric cancer patients with bacteremia treated at the Pediatric Department of Yeungnam University Hospital. We investigated and analyzed the causative organisms and the antibiotic sensitivity test results by reviewing the records of the microbiologically proven positive blood culture results. Results : The incidence of bacteremia in pediatric cancer patients in this study was 5.7 percent (58 episodes out of 1,022 occasions of blood cultures). Gram-positive organisms were isolated more often than gram-negative organisms (63.8 percent vs 36.2 percent) in the following order : Staphylococcus epidermidis (37.9 percent), Staphylococcus aureus (17.3 percent), Escherichia coli (12 percent), Streptococcus (8.6 percent), Enterobacter (6.9 percent), Klesiella (6.9 percent), Serratia (3.5 percent), Acinetobacter (3.5 percent), Proteus (1.7 percent) and Morganella morganii (1.7 percent). In antibiotic sensitivity tests, only six of 37 isolates (16 percent) of gram positive bacteria were sensitive to penicillin and 15 of 37 isolates (40 percent) were sensitive to oxacillin. All except one Staphylococcus aureus were sensitive to vancomycin and all except one Staphylococcus epidermidis were sensitive to teicoplanin among 37 isolates of gram positive bacteria. In the case of gram negative bacteria, two of 21 isolates (10 percent) and four of 21 isolates (19 percent) were sensitive to cefotaxime and ceftazidime, respectively. Only six of 21 isolates (29 percent) were sensitive to aminoglycoside, but all 21 isolates (100 percent) were sensitive to imipenem. All seven isolates tested after the year 2000 were sensitive to meropenem. Conclusion : In conclusion, we should choose the proper antimicrobials in treating pediatric cancer patients with suspected bacteremia, reflecting the increasing episodes of gram positive bacteremia and polymicrobial resistance of gram positive and negative organisms.

Response of Soil Microbial Communities to Different Farming Systems for Upland Soybean Cultivation

Lee, Young-Han,Kim, Hoon The Korean Society for Applied Biological Chemistr 2011 Applied Biological Chemistry (Appl Biol Chem) Vol.54 No.3

Organic farming for sustainable agriculture depends on the effects of microbial activities in the soil. The present study evaluated seasonal changes in the soil microbial communities by fatty acid methyl ester (FAME) in an organic farming system (OFS) with rye rotation cropping management compared to those in a conventional farming system (CFS) with chemical fertilizers, pesticide and herbicide from May 2009 to October 2010 in an upland field. Concentrations of total bacteria, Gram-negative bacteria, Gram-positive bacteria, fungi, and arbuscular mycorrhizal fungi in the OFS were significantly higher than those in the CFS during soybean-growing stages (p<0.001). The communities of total bacteria, Gram-negative bacteria, Gram-positive bacteria, actinomycetes and arbuscular mycorrhizal fungi in the CFS soils were significantly higher than those in the OFS soils (p<0.001), whereas the community of fungi was significantly lower in the CFS soils than in the OFS soils (p<0.05) due to decreasing Gram-positive bacteria. OFS had a significantly lower ratio of $cy19:0/18:1{\omega}7c$ compared with CFS, indicating microbial stress decreased (p<0.001). In principal component analyses of soil microbial communities, our findings suggested that Gram-positive bacteria should be considered as potential factor responsible for the clear microbial community differentiation observed between OFS and CFS in upland fields.

The Cell and Genetic Characteristics of Slime Forming Bacteria on Antibiotic Resistance in the Paper-making Process

Eun-HeeKim,오정수,강규영,조병묵 한국목재공학회 2002 목재공학 Vol.30 No.3

The seven strains, Pseudomonas paucimobilis, Pseudomonas cepacia, Staphylococcus auricularis, Staphylococcus saprophyticus, Acidovorax spp., Acinetobacter calcoaceticus, and Actinobacillus capsulatus were tested with three slimicides. Most of the tested bacteria were inhibited with slimicide K (an isothiazolin based compound), even at its low concentration, except for Actinobacillus capsulatus and Staphylococcus auricularis. Both slimicides B (an organic bromine based compound) and S (aldehydes) also couldn't prevent these two strains even at their highest concentration. Five different sizes of plasmid DNA were isolated from Actinobacillus capsulatus. Staphylococcus auricularis, a gram-positive bacteria, showed the slimy substances around its cell distinctively. The results suggest that two strains, Actinobacillus capsulatus, Staphylococcus auricularis, have presumably developed a resistance to the slimicide, by plasmid DNA or slimy substance.Our findings also suggest that not only gram-negative bacteria, but also gram-positive bacteria should not be neglected.

Response of Soil Microbial Communities to Different Farming Systems for Upland Soybean Cultivation

( Young Han Lee ),( Hoon Kim ) 한국응용생명화학회 2011 Applied Biological Chemistry (Appl Biol Chem) Vol.54 No.3

Organic farming for sustainable agriculture depends on the effects of microbial activities in the soil. The present study evaluated seasonal changes in the soil microbial communities by fatty acid methyl ester (FAME) in an organic farming system (OFS) with rye rotation cropping management compared to those in a conventional farming system (CFS) with chemical fertilizers, pesticide and herbicide from May 2009 to October 2010 in an upland field. Concentrations of total bacteria, Gram-negative bacteria, Gram-positive bacteria, fungi, and arbuscular mycorrhizal fungi in the OFS were significantly higher than those in the CFS during soybean-growing stages (p<0.001). The communities of total bacteria, Gram-negative bacteria, Gram-positive bacteria, actinomycetes and arbuscular mycorrhizal fungi in the CFS soils were significantly higher than those in the OFS soils (p<0.001), whereas the community of fungi was significantly lower in the CFS soils than in the OFS soils (p<0.05) due to decreasing Gram-positive bacteria. OFS had a significantly lower ratio of cy19:0/18:1ω7c compared with CFS, indicating microbial stress decreased (p<0.001). In principal component analyses of soil microbial communities, our findings suggested that Gram-positive bacteria should be considered as potential factor responsible for the clear microbial community differentiation observed between OFS and CFS in upland fields.

Gram-Positive Bacteria Specific Properties of Silybin Derived from Silybum marianum

DongGunLee,HyungKeunKim,YoonkyungPark,박성철,Eun-RhanWoo,정혜광,Kyung-SooHahm 대한약학회 2003 Archives of Pharmacal Research Vol.26 No.8

Silybin has a potent antibacterial activity, more potent than silymarin II, against gram-positive bacteria without hemolytic activity, whereas it has no antimicrobial activity against gram-negative bacteria or fungi. The mode of action of silybin against the gram-positive bacterial cell was examined by investigating the change in plasma membrane dynamics of bacterial cells using 1,6-diphenyl-1,3,5-hextriene (DPH) as a membrane probe and by assessing the inhibition of macromolecular synthesis using radiolabeled incorporation assay. The results showed that silybin inhibited RNA and protein synthesis on gram-positive bacteria.

Gram-Positive Bacteria Specific Properties of Silybin Derived from Silybum marianum

Lee, Dong-Gun,Kim, Hyung-Keun,Park, Yoon-Kyung,Park, Seong-Cheol,Woo, Eun-Rhan,Jeong, Hye-Gwang,Hahm, Kyung-Soo The Pharmaceutical Society of Korea 2003 Archives of Pharmacal Research Vol.26 No.8

Silybin has a potent antibacterial activity, more potent than silymarin II, against gram-positive bacteria without hemolytic activity, whereas it has no antimicrobial activity against gram-negative bacteria or fungi. The mode of action of silybin against the gram-positive bacterial cell was examined by investigating the change in plasma membrane dynamics of bacterial cells using 1 ,6-diphenyl-1,3,5-hextriene (DPH) as a membrane probe and by assessing the inhibition of macromolecular synthesis using radiolabeled incorporation assay. The results showed that silybin inhibited RNA and protein synthesis on gram-positive bacteria.

Crystal Structure of DsbA from Corynebacterium diphtheriae and Its Functional Implications for CueP in Gram-Positive Bacteria

엄시현,김진식,송새미,김남아,정성훈,하남출 한국분자세포생물학회 2015 Molecules and cells Vol.38 No.8

In Gram-negative bacteria in the periplasmic space, the dimeric thioredoxin-fold protein DsbC isomerizes and reduces incorrect disulfide bonds of unfolded proteins, while the monomeric thioredoxin-fold protein DsbA introduces disulfide bonds in folding proteins. In the Gram-negative bacteria Salmonella enterica serovar Typhimurium, the reduced form of CueP scavenges the production of hydroxyl radicals in the copper-mediated Fenton reaction, and DsbC is responsible for keeping CueP in the reduced, active form. Some DsbA proteins fulfill the functions of DsbCs, which are not present in Gram-positive bacteria. In this study, we identified a DsbA homologous protein (CdDsbA) in the Corynebacterium diphtheriae genome and determined its crystal structure in the reduced condition at 1.5 Å resolution. CdDsbA consists of a monomeric thioredoxin-like fold with an inserted helical domain and unique N-terminal extended region. We confirmed that CdDsbA has disulfide bond isomerase/reductase activity, and we present evidence that the N-terminal extended region is not required for this activity and folding of the core DsbA-like domain. Furthermore, we found that CdDsbA could reduce CueP from C. diphtheriae.

Crystal Structure of DsbA from Corynebacterium diphtheriae and Its Functional Implications for CueP in Gram-Positive Bacteria

Um, Si-Hyeon,Kim, Jin-Sik,Song, Saemee,Kim, Nam Ah,Jeong, Seong Hoon,Ha, Nam-Chul Korean Society for Molecular and Cellular Biology 2015 Molecules and cells Vol.38 No.8

In Gram-negative bacteria in the periplasmic space, the dimeric thioredoxin-fold protein DsbC isomerizes and reduces incorrect disulfide bonds of unfolded proteins, while the monomeric thioredoxin-fold protein DsbA introduces disulfide bonds in folding proteins. In the Gram-negative bacteria Salmonella enterica serovar Typhimurium, the reduced form of CueP scavenges the production of hydroxyl radicals in the copper-mediated Fenton reaction, and DsbC is responsible for keeping CueP in the reduced, active form. Some DsbA proteins fulfill the functions of DsbCs, which are not present in Gram-positive bacteria. In this study, we identified a DsbA homologous protein (CdDsbA) in the Corynebacterium diphtheriae genome and determined its crystal structure in the reduced condition at $1.5{\AA}$ resolution. CdDsbA consists of a monomeric thioredoxin-like fold with an inserted helical domain and unique N-terminal extended region. We confirmed that CdDsbA has disulfide bond somerase/reductase activity, and we present evidence that the N-terminal extended region is not required for this activity and folding of the core DsbA-like domain. Furthermore, we found that CdDsbA could reduce CueP from C. diphtheriae.