• Journal of Life Science
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• v.28 no.11
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• pp.1301-1315
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• 2018

We purified an antimicrobial peptide from the acidified hemocyte extract of Mytilus coruscus by $C_{18}$ reversed-phase high-performance liquid chromatography (RP-HPLC). The peptide was 4041.866 Da based on matrix-assisted laser desorption ionization time-of-flight mass spectrophotometer (MALDI-TOF/MS) and the 25 amino acids of the N-terminus sequence were identified. Comparison of this sequence of the purified peptide with the N-terminus sequences of other antimicrobial peptides revealed 100% identity with the mytilin B precursor of Mytilus coruscus. We also identified a 312 bp open-reading frame (ORF) encoding 103 amino acids based on the obtained amino acid residues. The nucleotide sequence of this ORF and the amino acid sequence also revealed 100% identity with the mytilin B precursor of Mytilus coruscus. We synthesized two antimicrobial peptides with an alanine residue in the C-terminus, and designated them mytilin B1 and B2. These two antimicrobial peptides showed antimicrobial activity against gram-positive bacteria, including Bacillus cereus and Streptococcus parauberis (minimal effective concentration, MECs $41.6-89.7{\mu}g/ml$), gram-negative bacteria, including Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Providencia stuartii, Pseudomonas aeruginosa, and Vibrio ichthyoenteri (MECs $7.4-39.5{\mu}g/ml$), and the fungus Candida albicans (MECs $26.0-31.8{\mu}g/ml$). This antimicrobial activity was stable under heat and salt conditions. Furthermore, the peptides did not exhibit significant hemolytic activity or cytotoxic effects. These results suggest that mytilin B could be applied as alternative antibiotic agent, and they add to the understanding of the innate immunity of hard-shelled mussels.

• Annals of Clinical Microbiology
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• v.22 no.1
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• pp.9-13
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• 2019

Background: The isolation of carbapenemase-producing Enterobacteriaceae (CPE) has become increasingly common. Continuous surveillance for these organisms is essential because their infections are closely related to outbreaks of illness and are associated with high mortality rates. The aim of this study was to develop and evaluate multiplex PCR as a means of detecting several important CPE genes simultaneously. Methods: We aimed to develop a multiplex PCR that could detect seven CPE genes simultaneously. The multiplex PCR was composed of seven primer sets for the detection of KPC, IMP, VIM, NDM-1, GES, OXA-23, and OXA-48. We designed different PCR product sizes of at least 100 bp. We evaluated the performance of this new test using 69 CPE-positive clinical isolates. Also, we confirmed the specificity to rule out false-positive reactions by using 71 carbapenem-susceptible clinical strains. Results: A total of 69 CPE clinical isolates showed positive results and were correctly identified as KPC (N=14), IMP (N=13), OXA-23 (N=12), OXA-48 (N=11), VIM (N=9), GES (N=5), and NDM (N=5) by the multiplex PCR. All 71 carbapenem-susceptible clinical isolates, including Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa, showed negative results. Conclusion: This multiplex PCR can detect seven CPE genes at a time and will be useful in clinical laboratories.

• Korean Journal of Veterinary Service
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• v.35 no.4
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• pp.295-305
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• 2012

The aim of this study was to isolate microorganisms from half milk samples of dairy goats by California mastitis test (CMT) during the lactation period and to further investigate the susceptibility of isolated organisms to antimicrobial drugs. From a total of 235 half milk samples with CMT scores of 2 or above from 366 dairy goats distributed throughout Jeonnam province, microorganisms were isolated from 198 (83.5%) samples either singly (99.0%) or in combination (1.0%). The most prevalent microorganism was the coagulase-negative Staphylococcus spp., (44.4%, n=88) followed by Staphylococcus aureus (24.2%, n=48), Escherichia coli (11.1%, n=22) and Streptococcus spp. (7.6%, n=15). Isolated bacteria also included Bacillus spp. (2.5%, n=5), Pseudomonas spp. (2.5%, n=5), Micrococcus spp. (1.5%, n=3), Corynebacterium spp. (1.5%, n=3), Enterococcus facium (1.0%, n=2), Morganella morganii (0.5%, n=1) and Streptococcus agalactiae (0.5%, n=1). During the summer season, a high prevalence of all microorganisms were observed in which Staphylococcus spp. (30.8%), Escherichia coli (8.6%), and Streptococcus spp. (5.6%) were among the most prevalent bacteria isolated. Staphylococcus spp. was also shown to be high in the winter (21.7%). In most samples, the presence of bacterial pathogens in goat milk led to the increase in the total somatic cell count (SCC). Most of the half milk samples of dairy goats with bacterial contamination showed SCC of ${\geq}1{\times}10^6cells/ml$ (90.4%). Minor pathogens (11.4%) were more detected from milk samples with SCC of < $1{\times}10^6cells/ml$ than major pathogens (4.1%), while the major pathogens tended to be higher from samples with SCC of ${\geq}3{\times}10^6cells/ml$. Susceptibility of these bacteria to 12 antimicrobial agents was tested by the Kirby-Bauer disc diffusion method. Results indicated that more than 90% of bacteria isolated from CMT 2+ dairy goat half milk samples were susceptible to trimethoprim/sulfamethoxazole, amoxicillin/clavulanic, enrofloxacin and cephalothin while they were resistant to tetracycline (44.7%).

• Korean Journal of Environmental Agriculture
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• v.15 no.4
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• pp.399-405
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• 1996

In the culture medium, the three antifungal fractions against P. capsici were separated by Sephadex G-25 column chromatography and Silica-gel chromatography. The substance A in white powder and the substance B in sticky oil were isolated by ethyl acetate : acetone mixture(7 : 3), and the substance C in yellow powder was isolated by chloroform : ethyl acetate mixture(95 : 5). The crude extract by ethyl acetate from the culture medium acidified to pH 2 was known to inhibit completely the growth of P. capsici at the level of $50mgkg^{-1}$. The substance A and B were known to be effective above the level of $5mgkg^{-1}$, and the substance C was effective above the level of $1mgkg^{-1}$. However, at the level of $20mgkg^{-1}$, the efficiency was in the order of A>C>B. It is apparent on a pot-experiment scale that the three substances effectively control Phytophthora blight of the red-pepper plant grown in the soil inoculated with P. capsici.

• Tuberculosis and Respiratory Diseases
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• v.40 no.1
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• pp.29-34
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• 1993

Background: The development of the flexible fiberoptic broncoscope by Ikeda was an important technologic advance in the diagnosis and management of patients with pulmonary disease. But, cross contamination related to fiberoptic bronchoscope was reported in cases involving tubercle bacilli, MOTT and other agents. Therefore, cleaning and disinfecting of fiberoptic bronchoscope requires careful attention. Methods: From September 1991 to May 1992, medical records of all patients with positive culture for MOTT in bronchial washing specimens were reviewed. Also to evaluate bactericidal effect of 2% glutaraldehyde, culture was performed after inoculum of MOTT, Serratia marsescens and Pseudomonas aeruginosa to the disinfectant solution. Results: In 2% alkaline glutaraldehyde, MOTT was not survived only after 30 minute exposure, but P. aeruginosa and S. marsescens were rapidly inactivated with no survivors after exposure to 2% glutaraldehyde. Since vigorous mechanical cleansing and more than 30 minute of contact time within washing machine, no more outbreak was observed. Conclusions: It is also very important that bronchoscopes must be meticulously cleaned after each procedure and more than 30 minute exposure would be required for eradication of MOTT with 2% glutaraldehyde. However even the most strictly applied infection control measures cannot exclude contamination completly and clinicians have to stay alert to this possibility. Prompt detection of pseudoepidemics is possible if abrupt increase in isolation rates, especially if they involve unusual or generally nonpathogenic organisms, are readily recognized.

• Korean Journal Plant Pathology
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• v.3 no.3
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• pp.187-197
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• 1987

Bacteria and fungi antagonistic to Fusarium oxysporum f. sp. cucumerinum Owen were effectively isolated with each of modified Triple Layer Agar (TLA) technique from rhizosphere soil where cucumber had been grown healthily in plastic film house. Three predominant bacterial isolates selected were identified as Pseudomonas fluorescens, and P. putida, Serratia sp. and three fungal isolates were Gliocladium sp. Trichoderma harzianum, and T. viride. Antagonistic bacteria inhibited $26-45\%$ of germination and $41-56\%$ of germ tube elongation of microconidia of F. oxysporum f. sp. cucumerinum on Water Agar (WA). P. fluorescens was the strongest inhibitor. Several my co parasitisms were observed on dual culture of WA between antagonistic fungi and F. oxysporum f. sp. cucumerinum such as coiling, penetration, overgrowing, and lysis. Mycelial lysis of the pathogen was the most severe at pH 4.6, followed by 3.6, 5.6 and 6.6 of the medium in decreasing order. At pH 6.6, mycelia of the pathogen were not conspicuously damaged, however, the antagonistic fungi formed abundant chlamydospores especially Gliocladium sp. T. harzianum revealed the most excellent antagonism in vitro.

• Microbiology and Biotechnology Letters
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• v.34 no.3
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• pp.196-203
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• 2006

Three slime-forming lactic acid bacteria were isolated from Kimchi and shown to produce viscous exopolysaccharides (EPS) in sucrose media. The isolated strains, GJ2, C3 and C11, were identified as Leuconostoc kimchii, Leuconostoc citreum and Leuconostoc mesenteroides, respectively, by examining their metabolic characteristics and determining their 16S rDNA sequences. Leu. kimchii GJ2, Leu. citreum C3 and Leu. mesenteroides C11 exhibited high viability (maintained initial viable cell count of $10^8$ CFU/ml) in 0.05 M sodium phosphate buffer (pH 3.0) for 2 h, in artificial gastric juice for 2 h and in 0.3% oxgall for 24 h. When tested, Leu. kimchii GJ2, in particular, displayed antimicrobial activity against pathogenic microorganisms. Leu. kimchii GJ2, Leu. citreum C3 and Leu. mesenteroides C11 produced 21.49 g/l, 16.46 g/l and 22.98 g/l EPS, respectively, in sucrose (5%) medium. The amount of purified EPS extracted from Leu. kimchii GJ2, Leu. citreum C3 and Leu. mesenteroides C11 was 14.61 g/l, 7.73 g/l and 4.77 g/l, respectively. Although the EPS produced by Leu. kimchii GJ2, Leu. citreum C3 and Leu. mesenteroides C11 differed in viscosity, TLC and HPLC analysis revealed that each contained only one type of monosaccharide, glucose. The average molecular mass of EPS produced by Leu. kimchii GJ2 was 306,606 Da.

• Korean Journal of Veterinary Research
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• v.24 no.2
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• pp.149-162
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• 1984

The research was conducted(1) to confirm the agent(s) responsible for the antimicrobial activity contained in the fermented tomato juice with L. acidophilus(2) to extract and purify the antimicrobial agent(s)(3) to find the biological, physical and chemical properties of the agent(s). The following results were obtained and summarized as followings; 1. The agent responsible for the inhibitory activity was confirmed by both well assay method using fermented tomato juice with L. acidophilus and turbidimetric technique using the cell-free filtrate or neutralized filtrate of tomato acidohilus culture and found exerted antimicrobial agent other than lactic acid. 2. The procedures of purification : The isolation and purification of antimicrobial agent from the lyophilized acidophilus tomato culture were carried out by (1) methanol extraction (2) acetone extraction, (3) Sephadex G-50 gel filtration (4) paper chromatography and (5) thin layer chromatography. 3. The biological, physical and chemical properties of antimicrobial agent: The biological, physical, chemical properties of the purified antimicrobial agent were: (1) The antimicrobial activity was strong against test organisms; Bacillus subtilis(ATCC 6633), Escheichia coli(ATCC 25922), Staphylococcus aureus(ATCC 167), Pseudomonas fluorescens(KFCC 32394), Proteus vulgaris and Shigella dysenteriae. (2) The pH value of the agent was 2.0 and the inhibitory activity was lost when it was neutralized at 7.0 of pH and the agent was heat stable at $121^{\circ}C$ for 60 minutes. (3) The ultraviolet light absorption spectra of methanol-acetone extract and TLC fraction exhibited a maximum absorption at 260nm and 224nm respectively. (4) The most purified agent from TLC plate increased about 130-fold in activity. (5) The agent isolated from TLC plate was free from $H_2O_2$ or lactic acid. 4. Bioautographic assy: By means of bioautography of the agent on silica gel of TLC plate a strong inhibitory activity against B. subtilis was demonstrated. 5. Mass spectrometry: The agent obtained from TLC plate was analyzed by mass spectrometry which show the parent peak at m/e 264 suggesting the molecular weight of the compound and molecular group such as [$C_2H{^+}_4$], [CO], [CH=NH], [$C_3{H^}4_7$], [$\begin{array}{rcl}O\\{\parallel}\\CH_3-C\\\end{array}$], [$C_6-H{^+}_{11}$], [$C_5H{^+}_{11}$], [$\begin{array}{rcl}O\\{\parallel}\\C_5H_7-C^+\\\end{array}$] were suggested.

• Korean Journal of Microbiology
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• v.44 no.2
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• pp.130-134
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• 2008

Modern automated culture systems have increased the isolation rate of microorganisms and shortened the time to detection, reducing experimental errors in diagnosis of infecting agents. BacT/ALERT 3D system is based on the colorimetric detection of $CO_2$ produced by the growing microorganisms. In order to evaluate the efficiency of the detection system, sterility test were performed using 6 bacteria. With standard aerobic and anaerobic bottles containing the liquid media, both three aerobic bacteria (P. aeruginosa, M. luteus, B. subtilis) and a facultative bacterium S. aureus were detected up to 1 CFU in 31.44 hr. In addition, growth of anaerobic C. sporogenes was recognized up to 1 CFU in 15.96 hr. The slowly growing bacteria P. acnes was detected up to 10,000 CFU in 129.36 hr. In comparison with conventional culture method, BacT/ALERT 3D automated culture system was more sensitive and saved detection time up to$2\sim10$ hr. Therefore, this automated culture system enables to efficiently detect bacteria in clinical samples and biological medicines.

• Tuberculosis and Respiratory Diseases
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• v.72 no.4
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• pp.360-366
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• 2012

Background: Carbapenem-resistance is rapidly evolving among the pathogenic microbes in intensive care units (ICUs). This study aimed to determine annual trend of carbapenem-resistance in the ICU for 4 years, since the opening of a university-affiliated hospital in South Korea. Methods: From 2005 to 2008, microbial samples from consecutive 6,772 patients were screened in the ICU. Three hundred and ninety-seven patients (5.9%) and their first isolates of carbapenem-resistant pathogens were analyzed. Results: The percentage of patients infected with carbapenem-resistant organisms increased constantly during the initial three years (2.3% in 2005, 6.2% in 2006, 7.8% in 2007), then it declined to 6.5% in 2008. Acute Physiology and Chronic Health Evaluation (APACHE) III score at admission was $58.0{\pm}23.5$, the median length of the ICU stay was 37 days, and the mortality rate was 37.5%. The sampling sites were endotracheal suction (67%), catheterized urine (17%), wound (6%) and others (10%). Bacteria with carbapenem-resistance were Pseudomonas aeruginosa (247 isolates, 62%), Acinetobacter baumannii (117 isolates, 30%), Enterobacteriaceae (12 isolates, 3%), and others (21, 5%). Of note, peak isolation of carbapenem-resistant microorganisms in medical ICU was followed by the same epidemic at surgical ICU. Conclusion: Taken together, carbapenem-resistant pathogens are of growing concern in the ICU.