• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.1015-1025
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• 2015

The development of diverse polymyxin derivatives is needed to solve the toxicity and resistance problems of polymyxins. However, no platform has generated polymyxin derivatives by genetically engineering a polymyxin synthetase, which is a nonribosomal peptide synthetase. In this study, we present a two-step approach for the construction of engineered polymyxin synthetases by substituting the adenylation (A) domains of polymyxin A synthetase, which is encoded by the pmxABCDE gene cluster of Paenibacillus polymyxa E681. First, the seventh L-threonine-specific A-domain region in pmxA was substituted with the L-leucine-specific A-domain region obtained from P. polymyxa ATCC21830 to make polymyxin E synthetase, and then the sixth D-leucine-specific A-domain region (A_6-D-Leu-domain) was substituted with the D-phenylalanine-specific A-domain region (A_6-D-Phe-domain) obtained from P. polymyxa F4 to make polymyxin B synthetase. This step was performed in Escherichia coli on a pmxA-containing fosmid, using the lambda Red recombination system and the sacB gene as a counter-selectable marker. Next, the modified pmxA gene was fused to pmxBCDE on the chromosome of Bacillus subtilis BSK4dA, and the resulting recombinant strains BSK4-PB and BSK4-PE were confirmed to produce polymyxins B and E, respectively. We also succeeded in constructing the B. subtilis BSK4-PP strain, which produces polymyxin P, by singly substituting the A_6-D-Leu-domain with the A_6-D-Phe-domain. This is the first report in which polymyxin derivatives were generated by genetically engineering polymyxin synthetases. The two recombinant B. subtilis strains will be useful for improving the commercial production of polymyxins B and E, and they will facilitate the generation of novel polymyxin derivatives.

• The Journal of the Korean Society for Microbiology
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• v.6 no.1
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• pp.29-40
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• 1971

Various kinds of antibiotics are generally believed to have inhibitory effects on the antibody response. However, as polymyxin B which belongs to the cyclic polypeptide group of antibiotic was found to have some enhancing effects on the antibody response of rabbits to enterobacterial common antigen(CA) under specified conditions, experiments were carried out on this problem with the following results. 1. When mixture of polymyxin B and CA derived from Salmonella typhimurium(STM) was treated 30 minutes at $37^{\circ}C$ and injected three times into rabbits by intravenous route, the antibody response to CA was weaker than rabbits injected CA only. 2. Mixture of polymyxin B and CA showed a marked antibody production when injected into rabbits primed with small amounts of heat-extracted antigen of STM, while the injection of CA alone showed low titers of response. 3. Mixture of polymyxin B and heat-extracted CA-containing antigen of Escherichia coli 014 also showed a increased antibody production than CA alone in rabbits primed with antigen of STM. 4. The effect of polymyxin B appeared in different ways. This antibiotic did not enhance the CA antibody response in rabbits primed with small amounts of E. coli 0111 and 055, but enhance in rabbits primed with Shigella flexneri. 5. No enhancing effect on the antibody response was observed by polymyxin B in rabbits primed with CA. 6. No enhancing effect on the antibody response was also noted in rabbits primed with STM antigen in case polymyxin B and CA were administered simultaneously but in veins of different places. 7. Bacitracin did not enhance the CA antibody response in primed rabbits with STM antigen, but neomycin slightly enhance the response. 8. Lipopolysaccharide showed no priming effect on the CA antibody response, and no enhancement of the CA antibody response in rabbits printed with STM. 9. The priming effect of STM antigen against CA antibody response was very weak as compared with the effect of CA derived from STM antigen.

• Korean Journal of Microbiology
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• v.45 no.2
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• pp.208-214
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• 2009

To avoid ambiguity in counting the number of colony, about 1,500 of colonies grown on B. cereus selective agar plates were grouped into 12 types by morphological difference and then identified by biochemical and 16S rDNA nucleotide sequence. Among them, seven colony types with 11 to 15 mm diameters of halo were identified as B. cereus or B. cereus subsp. cytotoxis. Five mm sized colonies with no halo, which have not been considered as B. cereus according to the manufacturer's manual, were identified as B. cereus. A colony type with double halos of only 6 mm in diameter was also B. cereus. Other three types were proven to be Enterococcus sp., Brevibacillus sp., and B. subtilis, respectively. PCR results showed that only 9 types that are identified as B. cereus strains harbor at least one of B. cereus toxin genes.

• The Journal of Korean Medicine
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• v.19 no.2
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• pp.36-49
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• 1998

HERBA SAURURI (HS) has been known to use antiinflammatory drug. To investigated the mechanism of HS-induced NO synthesis, I evaluated the ability of protein kinase C (PKC) inhibitors such as staurosporine (STSN) or polyymyxin B to block HS-induced effects. HS alone had only a small effect, whereas in combination with $rIFN-{\gamma}$, markedly increased NO synthesis in a dose dependent manner. STSN and polymyxin B decreased NO synthesis, which had been induced by $rIFN-{\gamma}$, plus HS. Furthermore, prolonged incubation of the cells with phorbol ester, which down-regulates PKC activity abolished synergistic cooperative effect of HS with $rIFN-{\gamma}$ on NO synthesis. STSN and Polymyxin B potently inhibited HS-induced $TNF-{\alpha}$ secretion by $rIFN-{\gamma}$ plus HS. However, $rIFN-{\gamma}$ plus $TNF-{\alpha}-induced$ NO synthesis was not blocked by STSN or polymyxin B. On the other hand, tyrosine kinase inhibitor, genistein, blocked the NO synthesis and $TNF-{\alpha}$ secretion by $rIFN-{\gamma}$ plus HS. In conlusion, the present results strongly suggest that the capacity of HS to increase NO synthesis from $rIFN-{\gamma}-primed$ macrophages is the result of HS-induced $TNF-{\alpha}$ secretion via the signal transduction pathway of PKC and tyrosine kinase.

• Journal of Life Science
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• v.14 no.4
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• pp.664-669
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• 2004

The contamination of Bacillus cereus was investigated in 240 RTE (ready-to-eat) food samples including 118 seafoods, 82 Korean packaged meals and 40 other RTE foods. Many B. cereus presumptive strains were isolated from the enrichment culture in Tryptic Soy Broth (TSB) added polymyxin, followed by selective culture in Mannitol Egg Yolk Polymyxin (MYP) agar and Gram staining. A total of 36 strains (16 in seafoods, 17 in Korean pack-aged meals and 3 in other RTE foods) were identified as B. cereus by the analysis of 61 biochemical tests of the API 50CHB/20E system test and supplementary tests of $\beta$-hemolysis, rhizoid growth, motility and oxidase activity. The 28 strains out of 36 B. cereus isolates produced diarrhoeal enter-otoxin in Brain Heart Infusion (BHI) broth. All isolates were resistant to ampicillin and penicillin antibiotics, and most of them were susceptible to gentamicin, vancomycin, bacitracin, chloram-phenicol, kanamycin and streptomycin. The growth of B. cereus was affected by environmental temperature and incubation time. Culture with temperature under 1$0^{\circ}C$ effectively restricted the growth of B. cereus.

• Journal of Food Hygiene and Safety
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• v.35 no.4
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• pp.319-325
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• 2020

Widespread consumption of fresh-cut vegetables without cooking results in ingestion of major foodborne pathogens including Bacillus cereus. In this study, we aimed to develop a method to rapidly detect B. cereus in fresh-cut vegetables by combining commercial PCR analysis with enrichment of the pathogenic levels. A mixture of B. cereus strains (KCTC1013, KCTC1014, KCTC1092, KCTC1094, and KCTC3624) was inoculated on the surface of fresh-cut cabbage lettuce (20 g) and baby leafy vegetables (10 g) to concentration 1, 2, 3, 4, and 5 log CFU/g. Eighty milliliters of TSB with 0.15% polymyxin B was used for cabbage lettuce, and 90 mL of medium was used for baby leafy vegetables and incubated at 42℃ for 0, 2, 3, 4, 5, 6, and 7 h. One milliliter of the enriched media was plated on mannitol-egg yolk-polymyxin agar for quantification, and another 1 mL was used for DNA extraction for PCR analysis. Additionally, the minimum number of sub-samples to be tested from a pack of fresh-cut vegetable samples was determined using 5 sub-samples. The results from this study showed that for detecting B. cereus in fresh-cut cabbage lettuce, 3, 4, 5, 6, and 7 h enrichment were required to at least detect 5, 4, 3, 2, and 1 log CFU/g of B. cereus, respectively. B. cereus in fresh-cut baby leafy vegetables could be detected after 2, 3, 4, 5, and 6 h of enrichment at 5, 4, 3, 2, and 1 log CFU/g, respectively, using a combination of enrichment and PCR analysis. To determine if a pack of fresh-cut vegetable is positive, the minimum number of sub-samples should be 3. These results can be used to develop a rapid detection method to semi-quantify B. cereus in fresh-cut vegetable samples combining enrichment and PCR.

• The Korean Journal of Pharmacology
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• v.32 no.1
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• pp.31-37
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• 1996

The protective effect of 'ischemic preconditioning (IP)'on ischemia-reperfusion injury of heart has been reported in various animal species, but the mechanism is unclear. In an attempt to elucidate the mechanism of IP, we examined the effects of blockers against adenosine and protein kinase C in preconditioned heart of rat. The hearts perfused with oxygen-saturated Krebs-Henseleit solution by Langendorff method were exposed to 30 min global ischemia followed by 20 min reperfusion. IP was performed with three episodes of 5 min ischcmia and 5 min reperfusion just before ischemia-reperfusion. IP prevented the depression of contractile function and the myocardial contracture in the ischemic-reperfused heart and reduced the release of lactate dehydrogenase during the reperfusion period. Polymyxin B, chelerythrine and colchicine, PKC inhibitors, attenuated almost completely the anti-ischemic effect of IP, while adenosine receptor antagonists did not. These results indicate that PKC may be a crucial intracellular mediator in anti-ischemic action of IP in ischemic-reperfused rat heart, while adenosine may not be involved in the mechanism of IP.

• Korean Journal of Food Science and Technology
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• v.53 no.6
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• pp.815-818
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• 2021

The objective of this study was to develop a differential medium with improved selectivity for the isolation of Bacillus cereus. Mannitol egg yolk polymyxin medium supplemented with D-galactose allowed the differentiation of diarrheal- and emetic-type B. cereus through pH monitoring. The pH of the medium decreased significantly when incubating the emetic-type B. cereus, whereas the pH change was not significant when incubating the diarrheal-type. The addition of pH indicators, such as methyl red and phenol red, to the medium allowed visual differentiation between diarrheal- and emetic-type B. cereus. A solid agar medium was also developed by optimizing the concentrations of medium components such as monosaccharides, agar, egg yolk enrichment, pH indicators, and antibiotics. This study indicates the possibility of applying selective media for the differentiation of diarrheal- and emetic-type B. cereus.

• Journal of Dairy Science and Biotechnology
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• v.33 no.2
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• pp.93-102
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• 2015

The aim of this study was to investigate the physiological characteristics and immunomodulatory activity of Lactobacillus fermentum 450 isolated from raw milk. L. fermentum 450 showed optimum growth at $40^{\circ}C$ and exhibited immunomodulatory effects on $interleukin-1{\alpha}$, tumor necrosis $factor-{\alpha}$, and nitrous oxide at concentrations of >2,500 pg/mL, >2,000 pg/mL, and $11.55{\pm}2.95{\mu}M$, respectively. Of the 16 antibiotics tested, L. fermentum 450 exhibited the highest sensitivity to rifampicin, followed by penicillin-G, and the highest resistance to kanamycin, followed by neomycin and polymyxin B. The strain showed high acid phosphatase activity and was comparatively tolerant to bile juice and acid. Moreover, the strain displayed high resistance to Salmonella Typhimurium (63.86%). These results demonstrate that L. fermentum 450 has potential for use as a probiotic with immunomodulatory activity.

• Journal of Veterinary Clinics
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• v.27 no.1
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• pp.6-10
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• 2010

Bacterial pathogens were isolated from dogs with urinary tract infection (UTI) in local animal hospitals between August 2003 and December 2009. Bacteria were isolated from urine of 47 dogs. The isolated pathogens were Escherichia coli (n = 27), Streptococcus spp. (n = 7), Staphylococcus spp. (n = 5), Enterobacter spp. (n = 3), Proteus spp. (n = 2), other species were 3 strains, respectively. E. coli were susceptible to imimpenem, polymyxin B, amikacin, cephalosporins, aztreonam, amoxicillin clavulate, cephalosporins, tricarcillin, and amoxicillin clavulate, while were resistant bacitracin, erythromycin, lincomycin, oxacillin, penicillin, and novobiocin. Streptococcus spp. were susceptible to bacitracin, imimpenem, and trimethoprime-sulfa, while were highly resistant amikacin, cefotaxim, cefoxitin, cloxacillin, gentamicin, lincomycin, oxacillin, penicillin, streptomycin, and tobramycin. Staphylococcus spp. were susceptible to cefoxitin, doxycycline, enrofloxacin, imimpenem, and tobramycin, but were resistant aztreonam and tetracycline.