• Title/Summary/Keyword: bacterial culture

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Isolation and Characterization of Bacillus Strain as a Potential Biocontrol Agent (환경친화적 미생물농약으로서의 잠재성을 가진 세균의 분리 및 특성)

  • Lee, Ye-Ram;Lee, Sang-Mee;Jang, Eun-Young;Hong, Chang-Oh;Kim, Keun-Ki;Park, Hyean-Cheal;g Lee, Sang-Mon;Kim, Young-Gyun;Son, Hong-Joo
    • Journal of Life Science
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    • v.25 no.12
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    • pp.1408-1414
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    • 2015
  • In this study, to retain a stable bacterial inoculant, Bacillus strains showing antifungal activity were screened. The improved production, antifungal mechanism, and stability of the antifungal metabolite by a selected strain, AF4, a potent antagonist against phytopathogenic Botrytis cinerea, were also investigated. The AF4 strain was isolated from rhizospheric soil of hot pepper and identified as Bacillus subtilis by phenotypic characters and 16S rRNA gene analysis. Strain AF4 did not produce antifungal activity in the absence of a nitrogen source and produced antifungal activity at a broad range of temperatures (25-40℃) and pH (7-10). Optimal carbon and nitrogen sources for the production of antifungal activity were glycerol and casein, respectively. Under improved conditions, the maximum antifungal activity was 140±3 AU/ml, which was higher than in the basal medium. Photomicrographs of strain AF4-treated B. cinerea showed morphological abnormalities of fungal mycelia, demonstrating the role of the antifungal metabolite. The B. subtilis AF4 culture exhibited broad antifungal activity against several phytopathogenic fungi. The antifungal activity was heat-, pH-, solvent-, and protease-stable, indicating its nonproteinous nature. These results suggest that B. subtilis AF4 is a potential candidate for the control of phytopathogenic fungi-derived plant diseases.

Causative Agents and Antimicrobial Sensitivity of Neonatal Sepsis : Ten-year Experience in One Neonatal Intensive Care Unit (단일 신생아중환자실에서 경험한 10년간의 신생아 패혈증의 원인균 및 항생제 감수성 변화)

  • Park, Hye-Won;Lim, Gin-A;Koo, So-Eun;Lee, Byong-Sop;Kim, Ki-Soo;Pi, Soo-Young;Kim, Ai-Rhan
    • Neonatal Medicine
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    • v.16 no.2
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    • pp.172-181
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    • 2009
  • Purpose: To identify trends in causative bacterial organisms for neonatal sepsis and antimicrobial susceptibilities over 10 years in one neonatal intensive care unit. Methods: We retrospectively reviewed the cases of culture-proven neonatal sepsis between January 1998 and December 2007. The 10-year period was divided into two phases (phase I, 1998-2002; phase II, 2003-2007) to distinguish the differences during the entire period. Results: Total 350 episodes of neonatal sepsis were identified in 315 neonates. The common pathogens of early-onset sepsis were S. epidermidis, S. aureus, P. aeruginosa, and E. cloacae in phase I, and S. epidermidis and E. cloacae in phase II. In cases of late-onset sepsis, coagulase negative Staphylococcus, S. aureus, and K. pneumoniae were isolated frequently in both phases. The incidence of sepsis caused by multi-drug resistant organisms decreased with strict infection control. Gram positive organisms showed 0-20% susceptibility to penicillin, ampicillin, and cefotaxime in both phases. Sensitivity to amikacin for Enterobacter spp. increased, whereas P. aeruginosa showed decreased sensitivity in phase II. Between 50% and 60% of other gram negative bacteria, except P. aeruginosa, were susceptible to cefotaxime in phase II in contrast to phase I. Greater than 80% of gram negative bacteria were sensitive to imipenem except P. aeruginosa and ciprofloxacin in both phases. Conclusion: The trend in causative microorganisms and antimicrobial susceptibilities can be used as a guideline for selection of appropriate antibiotics. A particular attention should be paid to infection control, especially to reduce sepsis caused by multi-drug resistant organisms.

Identification of Antagonistic Bacteria, Pseudomonas aurantiaca YC4963 to Colletotri­chum orbiculare Causing Anthracnose of Cucumber and Production of the Antibiotic Phenazine-l-carboxylic acid (Colletotrichum orbiculare에 대한 길항세균 Pseudomonas aurantiaca YC4963의 분리 동정 및 항균물질 Phenazine-1-carboxylic acid의 생산)

  • Chae Hee-Jung;Kim Rumi;Moon Surk-Sik;Ahn Jong-Woong;Chung Young-Ryun
    • Korean Journal of Microbiology
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    • v.40 no.4
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    • pp.342-347
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    • 2004
  • A bacterial strain YC4963 with antifungal activity against Colletotrichum orbiculare, a causal organism of cucumber anthracnose was isolated from the rhizosphere soil of Siegesbeckia pubescens Makino in Korea. Based on physiological and biochemical characteristics and 16S ribosomal DNA sequence analysis, the bac­terial strain was identified as Pseudomonas aurantiaca. The bacteria also inhibited mycelial growth of several plant fungal pathogens such as Botrytis cinerea, Fusarium oxysporum and Rhizoctonia solani on PDA and 0.1 TSA media. The antifungal activity was found from the culture filtrate of this isolate and the active compound was quantitatively bound to XAD adsorption resin. The antibiotic compound was purified and identified as phenazine-l-carboxylic acid on the basis of combined spectral and chemical analyses data. This is the first report on the production of phenazine-l-carboxylic acid by Pseudomonas aurantiaca.

Detection of Campylobacter jejuni in food and poultry visors using immunomagnetic separation and microtitre hybridization

  • Simard, Ronald-E.
    • Proceedings of the Korean Society of Fisheries Technology Conference
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    • 2000.05a
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    • pp.71-73
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    • 2000
  • Campylobacter jejuni is most frequently identified cause of cause of acute diarrhoeal infections in developeed countries, exceeding rates of illness caused by both salmonella and shigilla(Skirrow, 1990 ; Lior 1994). Previous studies on campylobacter jejuni contamination of commercial broiler carcasses in u.s.(Stern, 1992). Most cases of the disease result from indirect transmission of Campylobactor from animals via milk, water and meat. In addition to Campylobactor jejuni. the closely relates species Campylobactor coli and Campylobactor lari have also been implicated as agents of gastroenteritis in humans. Campylobactor coli represented only approximately 3% of the Campylobactor isolates from patients with Campylobactor enteritis(Griffiths and Park, 1990) whereas Campylobactor coli is mainly isolated from pork(Lmmerding et al., 1988). Campylobactor jejuni has also been isolated from cases of bacteremia, appendicitis and, recently, has been associated with Guillai-Barre syndrome(Allos and Blaser, 1994; von Wulffen et al., 1994; Phillips, 1995). Studies in volunteers indicated that the infectious dose for Campylobactor jejuni is low(about 500 organisms)(Robinson, 1981). The methods traditionally used to detect Campylobactor ssp. in food require at least two days of incubation in an enrichment broth followed by plating and two days of incubation on complex culture media containing many antibiotics(Goossens and Butzler, 1992). Finnaly, several biochemical tests must be done to confirm the indentification at the species level. Therfore, sensitive and specific methods for the detection of small numbers of Campylobactor cells in food are needed. Polymerase chain reaction(PCR) assays targeting specific DNA sequences have been developed for the detection of Campylobactor(Giesendorf and Quint, 1995; Hemandex et al., 1995; Winter and Slavidk, 1995). In most cases, a short enrichment step is needed to enhance the sensitivity of the assay prior to detection by PCR as the number of bacteria in the food products is low in comparison with those found in dinical samples, and because the complex composition of food matrices can hinder the PCR and lower its sensitivity. However, these PCR systems are technically demanding to carry out and cumbersome when processing a large number of samples simutaneously. In this paper, an immunomagnetic method to concentrate Campylobactor cells present in food or clinical samples after an enrichment step is described. To detect specifically the thermophilic Campylobactor. a monoclonal antibody was adsorbed on the surface of the magnetic beads which react against a major porin of 45kDa present on the surface of the cells(Huyer et al., 1986). After this partial purification and concentration step, detection of bound cells was achieved using a simple, inexpensive microtitre plate-based hybridization system. We examined two alternative detection systems, one specific for thermophilic Campylobactor based on the detection of 23S rRNA using an immobilized DNA probe. The second system is less specific but more sensitive because of the high copy number of the rRNA present in bacterial cell($10^3-10^4$). By using specific immunomagnetic beads against thermophilic Campylobactor, it was possible to concentrate these cells from a heterogeneous media and obtain highly specific hybridization reactions with good sensitivity. There are several advantages in using microtitre plates instead of filter membranes or other matrices for hybridization techniques. Microtitre plates are much easier to handle than filter membranes during the adsorption, washing, hybridization and detection steps, and their use faciilitates the simultanuous analysis of multiple sample. Here we report on the use of a very simple detection procedure based on a monoclonal anti-RNA-DNA hybrid antibody(Fliss et al., 1999) for detection of the RNA-DNA hybrids formed in the wells.

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Measurement of the Levels of IgG Subclasses Reactive to Salmonella typhi in the Sera of Patients with Typhoid Fever (장티푸스환자의 혈청내 Salmonella typhi에 대한 IgG subclass항체의 분포)

  • Kim, Young-Jung;Hwang, Eung-Soo;Kang, Jae-Seung;Cha, Chang-Yong;Chang, Woo-Hyun;Kim, Yoon-Won;Cho, Min-Ki;Min, Chang-Hong
    • The Journal of the Korean Society for Microbiology
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    • v.21 no.4
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    • pp.447-453
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    • 1986
  • To diagnose the typhoid fever rapidly and accurately in clinically suspected patients, the levels of IgG subclass antibody were measured by enzyme-linked immunosorbent assay(ELISA). With symptom, blood culture and agglutination test, tested persons were categorized into 6 groups as typhoid fever, FUO, paratyphi A or B, other bacterial infctions, cancers, and control. ELISA was performed on the polyvinyl chloride plates coated with killed whole cell($10^8\;cell/ml$) of S. typhi 0901W by poly-L-lysine applied as binding substance (and polyvinyl chloride as solid phase). The distribution of the level of IgG subclass antibodies in each group was analyzed and compared with other groups. The results obtained were summarized as follow: 1. The optimal dilution of the sera from patients with typhoid fever was 1:160, and those of the sheep anti-human IgG subclass and the peroxidase conjugated rabbit anti-sheep IgG were 1:4000 and 1:5000, respectively. 2. The absorbance levels of IgG subclass in the sera of typhoid fever patients were as follows; a) IgG1 value is $0.439{\pm}0.110$ b) IgG2 value is $0.416{\pm}0.165$ c) IgG3 value is $0.449{\pm}0.145$ d) IgG4 value is $0.525{\pm}0.154$ IgG subclass levels in the sera of typhoid patients were much higher than in control group and patient with paratyphi A or B as well as other infectious diseases. The sensitivity and the specificity in differential diagnosis of typhoid fever and other febrile diseases were 92% and 79% in the assay of IgG1 respectively, whereas those in the assay of IgG2 were 97% and 72%, respectively (above absorbance 0.3). 3. The absorbance levels of IgG subclass in the serial sera of typhiod fever patients tend to decrease to the level of absorbance 0.3 in 10 months from the onset of illness. 4. The order of absorbance levels of IgG subclass in the serum of each group were typhoid fever, paratyphi A or B, other infectious diseases, control and cancer. 5. For the serodiagnosis of typhoid fever against other febrile diseases, the sensitivity and the specificity in the assay of IgG2 activity were 76% and 93% in absorbance 0.4, respectively. 6. In the distribution of the level of each IgG subclass in the sera of FUO patients which were suspected of typhoid fever, the positive rate was ranged from 36% to 82%. This suggest that more than 50% of FUO patients are caused by S. typhi.

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Characterization of Bacteriocin Produced from Isolated Strain of Bacillus sp. (Bacillus 속 분리주가 생산하는 박테리오신의 특성 조사)

  • Ham, Seung-Hee;Choi, Nack-Shick;Moon, Ja-Young;Baek, Sun-Hwa;Lee, Song-Min;Kang, Dae-Ook
    • Journal of Life Science
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    • v.27 no.2
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    • pp.202-210
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    • 2017
  • As an effort to find a potential biopreservative, we isolated bacterial strains producing bacteriocin from fermented foods. A strain was finally selected and characteristics of the bacteriocin were investigated. The selected strain was identified as Bacillus subtilis E9-1 based on the 16S rRNA gene analysis. The culture supernatant of B. subtilis E9-1 showed antimicrobial activity against Gram-positive bacteria. Subtilisin A, ${\alpha}$-chymotrypsin, trypsin and proteinase K inactivated the antimicrobial activity, which means its proteinaceous nature, a bacteriocin. The bacteriocin activity was fully retained at the pH range from 2.0 to 8.0 and stable at up to $100^{\circ}C$ for 60 min. Solvents such as ethanol, isopropanol and methanol had no effect on the antimicrobial activity at the concentration of 100% but acetone and acetonitrile reduced the activity at up to 100% concentration. Cell growth of four indicator strains was dramatically decreased in dose-dependent manner. Listeria monocytogenes was the most sensitive, but Enterococcus faecium was the most resistant. Bacillus cereus and Staphylococcus aureus showed the medium sensitivity. The bacteriocin showed its antimicrobial activity against B. cereus and L. monocytogenes via bactericidal action. The number of viable cells of L. monocytogenes started to reduce after addition of bacteriocin to the minced beef. The bacteriocin was purified through acetone concentration, gel filtration chromatography and RP-HPLC. The whole purification step led to a 6.82 fold increase in the specific activity and 6% yield of bacteriocin activity. The molecular weight of the purified bacteriocin was determined to be 3.3 kDa by MALDI-TOF/TOF mass spectrometry.

Quality characteristics of Korean-Uzbekistanis fermented soybean paste (우즈베키스탄 고려인 제조 장류의 품질특성)

  • Seo, Bo Young;Eom, Jeong Seon;Song, Jin;Baek, Sung Yeol;Kim, Jae Hyun;Park, Shin Young;Choi, Hye Sun
    • Food Science and Preservation
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    • v.22 no.3
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    • pp.458-463
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    • 2015
  • Many Koreans, who are resident in Uzbekistan and Central Asia, are still avid fans of the Korean traditional soybean paste. No research study has been conducted on the Korean-Uzbekistanis soybean paste. The purpose of this study was to investigate the quality characteristics of the Korean-Uzbekistanis fermented soybean paste. Nine kinds of soybean pastes prepared by Korean-Uzbekistanis were collected, and their physiochemical properties (protease activity, amylase activity, amino type nitrogen content, reducing-sugar contents, and aerobic bacterial count) have been analyzed. The Korean-Uzbekistanis' fermented soybean paste (KU-SP) showed higher protease activity than the Korean fermented soybean paste (K-SP). The protease activities of KU-SP B, D, and H were 832, 807 and 630 unit/g, respectively, which were significantly different from others. In addition, the content of amino-type nitrogen in KU-SP B, D, and H were 777 mg%, 686 mg%, and 705 mg%, respectively. In the meanwhile, ${\alpha}$-amylase activities showed wide ranges starting from 10.82 to 47.98 unit/g. The KU-SP values, except for the KU-SP C, have shown higher activities than the K-SP. The reduced sugar content and totoal aerobic bacteria number were within the range of 0.55~3.43%/g and 7.24~8.79 log CFU/g, respectively. Finally, this research provided the basic data and information for the quality characteristics of commercial soybean paste prepared by the Korean-Uzbekistanis. These basic data can be useful for understanding the Korean food culture in Central Asia.

Effects of Beneficial Microorganisms and Mycorrhizal Fungus Colonized Rhizoplane on the Suppression of Root Rot Pathogen, Fusarium solani (근면 정착 유용 미생물과 균근균이 근부병원균, Fusarium solani의 발병억제에 미치는 영향)

  • Han, Ki-Don;Lee, Sang-Sun;Kim, Sung-Ho;Lee, Min-Woong
    • The Korean Journal of Mycology
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    • v.24 no.1 s.76
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    • pp.38-48
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    • 1996
  • The survival or colonization of beneficial organsisms and suppression of root rot of ginseng (Panax ginseng) by two distinct bacteria, Pseudomonas cepacia, Bacillus cereus and three mycorrhiza in pot soil were investigated and compared with uninoculated root. In separate inoculation, colonization of roots by P. cepacia was maintained at 6.25 (log cfu/g root) during growth for 10 days under pot culture conditions comparing to $5.62{\sim}6.19$ by mixed treatment with other organisms. Colonizations of P. cepacia were gradually decreased from 6.25 (log cfu/g root) in 10 days growth to 3.01 (log cfu/g root) in 270 days incubation period. This reduction was also investgated in combination treatments by B. cereus or F. solani. The numbers of Fusarium spp. were colonized high number in rhizosphere soil from 3.33 to 3.67 (log cfu/g root) in control within $10{\sim}60$days after treatment of pathogen F. solani, but it's numbers were markedly decreased in 270 days cultivation of plant from 3.33 to 1.02 (log cfu/g root) after treatment. In treatment of beneficial strains of P. cepacia and B. cereus, P. cepacia significantly suppressed the development of root rot from 4.3 in control to 1.2 in treatment, whereas B. cereus alone had no effect on the rate of disease suppression. The disease index $(1.8{\sim}2.3)$ in combination of two bacteria was reduced in plants inoculated with both P. cepacia and B. cereus comparing to the index (4.3) of control. As an effect of inoculation with mycorrhiza on disease suppression, suppression of root rot by F. solani was reduced to $1.2{\sim}1.6$ in disease index in treatment of Glomus albidum and Acaulospora longular comparing to 4.3 of control. In the treatment of bacterial strain P. cepacia and mycorrhizal fungus Glomus albidum, the disease suppression was apparent to 1.2 and 1.2 comparing to 4.3 of control in disease index respectively.

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Identification and Characterization of Lactobacillus salivarius subsp. salivarius from Korean Feces

  • Bae, Hyoung-Churl
    • Proceedings of the Korean Society for Food Science of Animal Resources Conference
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    • 2004.05a
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    • pp.89-119
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    • 2004
  • This study was conducted to isolate lactobacilli having probiotic characteristics to be used as health adjuncts with fermented milk products. Acid tolerant strains were selected in Lactobacilli MRS broth adjusted to pH 4.0 from 80 healthy persons (infants, children and adults). And bile tolerant strains were examined in Lactobacilli MRS broth in which 1.0% bile salt was added. By estimation above characteristics, the strains No. 27, which was isolated from adult feces, was selected and identified as Lactobacillus salivarius subsp. salivarius based on carbohydrate fermentation and 16S rDNA sequencing. It was used as a probiotic strain in fermented milk products. The pH of fermented milk decreased from pH 6.7 to 5.0 and titratable acidity increased from 0.3% to 1.0% by L. salivarius subsp. salivarius (isolation strain 20, 35, and 37), when incubated for 36 h at $37^{\circ}C$. The number of viable cell counts of fermented milk was maximized at this incubation condition. The SDS-PAGE evidenced no significant change of casein but distinct changes of whey protein were observed by isolated L. salivarius subsp. salivarius for titratable acidity being incubated by $0.9{\sim}1.0%$ at $37^{\circ}C$. All of the strains produced 83.43 to 131.96 mM of lactic acid and 5.39 to 26.85 mM of isobutyric acid in fermented products. The in vitro culture experiment was performed to evaluate ability to reduce cholesterol levels and antimicrobial activity in the growth medium. The selected L. salivarius subsp. salivarius reduced $23{\sim}38%$ of cholesterol content in lactobacilli MRS broth during bacterial growth for 24 hours at $37^{\circ}C$. All of the isolated L. salivarius subsp. salivarius had an excellent antibacterial activity with $15{\sim}25$ mm of inhibition zone to E. coli KCTC1039, S. enteritidis KCCM3313, S. typhimurium M-15, and S. typhimurium KCCM40253 when its pH had not been adjusted. Also, all of the isolated L. salivarius subsp. salivarius had partial inhibition zone to E. coli KCTC1039, E. coli KCTC0115 and S. enteritidis KCCM3313 when it had been adjusted to pH 5.7. The selected strains were determined to have resistances of twelve antibiotic. Strains 27 and 35 among the L. salivarius subsp. salivarius showed the highest resistance to the antibiotics. Purified ${\alpha}$-galactosidase was obtained by DEAE-Sephadex A-50 ion exchange chromatography, Mono-Q ion exchange chromatography and HPLC column chromatography from L. salivarius subsp. salivarius 27. The specific activity of the purified enzyme was 8,994 units/mg protein, representing an 17.09 folds purification of the original cell crude extract. The molecular weight of enzyme was identified about 53,000 dalton by 12% SDS-PAGE. Optimal temperature and pH for activity of this enzyme were $40^{\circ}C$ and 7.0 respectively. The enzyme was found to be stable between 25 and $50^{\circ}C$. ${\alpha}$-galactosidase activity was lost rapidly below pH 5.0 and above pH 9.0. This enzyme was liberated galactose from melibiose, raffinose, and stachyose, and also the hydrolysis rate of substrate was compound by HPLC. These results indicated that some of the L. salivarius subsp. salivarius (strain 27 and 35) are considered as effective probiotic strains with a potential for industrial applications, but the further study is needed to establish their use as probiotics in vivo.

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ISOLATION AND IDENTIFICATION OF BACTERIA FROM THE ROOT CANAL OF THE TEETH DIAGNOSED AS THE ACUTE PULPITIS AND ACUTE PERIAPICAL ABSCESS (급성 치수염 및 급성 치근단 농양의 치근관으로부터의 세균 분리 및 동정)

  • Lee, Yeon-Jae;Kim, Mi-Kwang;Hwang, Ho-Keel;Kook, Joong-Ki
    • Restorative Dentistry and Endodontics
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    • v.30 no.5
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    • pp.409-422
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    • 2005
  • The aim of this study was to identify the bacteria isolated from acute endodontic lesions by cell culture and 16S rDNA sequencing. The necrotic pulpal tissue was collected from 17 infected root canals, which were diagnosed as being either an acute pulpitis or acute periapical abscess. Samples were collected aseptically from the infected pulpal tissue of the infected root canals using a barbed broach and a paper point. The cut barbed broaches and paper points were transferred to an eppendorf tube containing 500 ul of 1 XPBS. The sample solution was briefly mixed and plated onto a BHI-agar plate containing $5\%$ sheep blood. The agar plates were incubated in a $37^{\circ}C$ anaerobic chamber for 7 days. The bacteria growing on the agar plate were identified by 16S rRNA coding gene (rDNA) cloning and sequencing at the species level. Among the 71 colonies grown on the agar plates, 56 strains survived and were identified. In dental caries involving the root canals, Streptococcus spp. were mainly isolated. Actinomyces, Clostridia, Bacteroides and Fusobacteria were isolated in the periapical lesion without dental caries. Interestingly, two new Actinomyces spp. (ChDC B639 and ChDC B631) were isolated in this study. These results showed that there was diversity among the species in endodontic lesions, This suggests that an endodontic infection is a mixed infection with a polymicrobial etiology. These results may offer the bacterial strains for pathogenesis studies related to an endodontic infection.