• 한국미생물·생명공학회지
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• 제49권3호
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• pp.305-315
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• 2021

The cotton leafworm, Spodoptera littoralis, is a major pest in Egypt and many countries worldwide, and causes heavy economic losses. As a result, management measures to control the spread of the worm are required. S. littoralis nucleopolyhedrovirus (SpliNPV) is one of the most promising bioagents for the efficient control of insect pests. In this study, a chitinase gene (chitA) of a 1.8 kb DNA fragment was cloned and fully characterized from SpliNPV-EG1, an Egyptian isolate. A sequence of 601 amino acids was deduced when the gene was completely sequenced with a predicted molecular mass of 67 kDa for the preprotein. Transcriptional analyses using reverse transcription polymerase chain reaction (RT-PCR) revealed that chitA transcripts were detected first at 12 h post infection (hpi) and remained detectable until 168 hpi, suggesting their transcriptional regulation from a putative late promoter motif. In addition, quantitative analysis using quantitative RT-PCR showed a steady increase of 7.86-fold at 12 hpi in chitA transcription levels, which increased up to 71.4-fold at 120 hpi. An approximately 50 kDa protein fragment with chitinolytic activity was purified from ChitA-induced bacterial culture and detected by western blotting with an anti-recombinant SpliNPV chitinase antibody. Moreover, purification of the expressed ChitA recombinant protein showed in vitro growth inhibition of two different fungi species, Fusarium solani and F. oxysporum, confirming that the enzyme assembly and activity was correct. The results supported the potential role and application of the SpliNPV-ChitA protein as a synergistic agent in agricultural fungal and pest control programs.

• Journal of Microbiology and Biotechnology
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• 제29권2호
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• pp.274-282
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• 2019

Mercury-resistant ($Hg^R$) bacteria were isolated from heavy metal polluted wastewater and soil collected near to tanneries of district Kasur, Pakistan. Bacterial isolates AZ-1, AZ-2 and AZ-3 showed resistance up to $40{\mu}g/ml$ against mercuric chloride ($HgCl_2$). 16S rDNA ribotyping and phylogenetic analysis were performed for the characterization of selected isolates as Bacillus sp. AZ-1 (KT270477), Bacillus cereus AZ-2 (KT270478) and Bacillus cereus AZ-3 (KT270479). Phylogenetic relationship on the basis of merA nucleotide sequence confirmed 51-100% homology with the corresponding region of the merA gene of already reported mercury-resistant Gram-positive bacteria. The merE gene involved in the transportation of elemental mercury ($Hg^0$) via cell membrane was cloned for the first time into pHLV vector and transformed in overexpressed C43(DE3) E. coli cells. The recombinant plasmid (pHLMerE) was expressed and the native MerE protein was obtained after thrombin cleavage by size exclusion chromatography (SEC). The purification of fusion/recombinant and native protein MerE by Ni-NTA column, dialysis and fast protein liquid chromatography (FPLC/SEC) involved unfolding/refolding techniques. A small-scale reservoir of wastewater containing $30{\mu}g/ml$ of $HgCl_2$ was designed to check the detoxification ability of selected strains. It resulted in 83% detoxification of mercury by B. cereus AZ-2 and B. cereus AZ-3, and 76% detoxification by Bacillus sp. AZ-1 respectively (p < 0.05).

• Asian-Australasian Journal of Animal Sciences
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• 제32권1호
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• pp.82-91
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• 2019

Objective: This experiment was conducted to compare the structure and composition of ruminal microorganisms in goats with high and low neutral detergent fibre (NDF) digestibility. Methods: Nineteen crossbred goats were used as experimental animals and fed the same total mixed rations during the 30-day pre-treatment and 6-day digestion trialperiods. All faeces were collected during the digestion period for measuring the NDF digestibility. Then, high and the low NDF digestibility individuals were chosen for the high NDF digestibility group (HFD) and low NDF digestibility group (LFD), respectively. Rumen contents were collected for total microbial DNA extraction. The V4 region of the bacterial 16S rRNA gene was amplified using universal primers of bacteria and sequenced using high-throughput sequencer. The sequences were mainly analysed by QIIME 1.8.0. Results: A total of 18,694 operational taxonomic units were obtained, within 81.98% belonged to bacteria, 6.64% belonged to archaea and 11.38% was unassigned microorganisms. Bacteroidetes, Firmicutes, and Proteobacteria were the predominant microbial phyla in both groups. At the genus level, the relative abundance of fifteen microorganisms were significantly higher (p<0.05) and six microorganisms were extremely significantly higher (p<0.01) in LFD than HFD. Overall, 176 core shared genera were identified in the two groups. The relative abundance of 2 phyla, 5 classes, 10 orders, 13 families and 15 genera had a negative correlation with NDF digestibility, but only the relative abundance of Pyramidobacter had a positive correlation with NDF digestibility. Conclusion: There were substantial differences in NDF digestibility among the individual goats, and the NDF digestibility had significant correlation with the relative abundance of some ruminal microorganisms.

• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.454-464
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• 2019

Salmonellosis is a highly contagious bacterial disease that threatens both human and poultry health. Tests that can detect Salmonella in the field are urgently required to facilitate disease control and for epidemiological investigations. Here, we combined loop-mediated isothermal amplification (LAMP) with a chromatographic lateral flow dipstick (LFD) to rapidly and accurately detect Salmonella. LAMP primers were designed to target the Salmonella invA gene. LAMP conditions were optimized by adjusting the ratio of inner to outer primers, $MgSO_4$ concentration, dNTP mix concentration, amplification temperature, and amplification time. We evaluated the specificity of our novel LAMP-LFD method using six Salmonella species and six related non-Salmonella strains. All six of the Salmonella strains, but none of the non-Salmonella strains, were amplified. LAMP-LFD was sensitive enough to detect concentrations of Salmonella enterica subsp. enterica serovar Pullorum genomic DNA as low as $89fg/{\mu}l$, which is 1,000 times more sensitive than conventional PCR. When artificially contaminated feed samples were analyzed, LAMP-LFD was also more sensitive than PCR. Finally, LAMP-LFD gave no false positives across 350 chicken anal swabs. Therefore, our novel LAMP-LFD assay was highly sensitive, specific, convenient, and fast, making it a valuable tool for the early diagnosis and monitoring of Salmonella infection in chickens.

• 한국미생물·생명공학회지
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• 제49권4호
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• pp.543-551
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• 2021

Nocardiopsis species produce bioactive compounds, such as antimicrobial and anti-cancer agents and toxins. However, no reports have described their anti-inflammatory and anti-fibrotic effects during nasal polyp (NP) formation. In this study, we investigated whether marine-derived bacterial Nocardiopsis sp. 13G027 exerts anti-inflammatory and anti-fibrotic effects on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and transforming growth factor (TGF)-β1-induced NP-derived fibroblasts (NPDFs). Nitric oxide (NO) and prostaglandin E₂ (PGE₂) levels were analyzed. Extract from Nocardiopsis sp. 13G027 significantly inhibited the upregulation of NO and PGE₂ in LPS-activated RAW 264.7 macrophages. The expression of mitogen-activated protein kinases (MAPKs) and protein kinase B (Akt/PKB) in LPS-induced RAW 264.7 macrophages was evaluated; smooth muscle alpha-actin (α-SMA), collagen type I (Col-1), and fibronectin also phosphorylated small mothers against decapentaplegic (SMAD) 2 and 3 in TGF-β1-stimulated NPDFs. The Nocardiopsis sp. 13G027 extract suppressed the phosphorylation of MAPKs and Akt and the DNA-binding activity of activator protein 1 (AP-1). The expression of pro-fibrotic components such as α-SMA, Col-1, fibronectin, and SMAD2/3 was inhibited in TGF-β1-exposed NPDFs. These findings suggest that Nocardiopsis sp. 13G027 has the potential to treat inflammatory disorders, such as NP formation.

• 치위생과학회지
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• 제22권2호
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• pp.99-107
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• 2022

Background: Obesity weakens acquired immunity and causes infection. This study aimed to investigate the relationship between the inflammatory markers in the gingival crevicular fluid and serum and periodontal bacteria in saliva through obesity control for 4 weeks. Methods: Forty-six subjects with a body mass index (BMI) of ≥23 kg/m² stayed in the camp for 4 weeks, followed by exercise and a low salt-low fat diet. Body size measurements, oral examinations, blood, saliva, and gingival crevicular fluid were collected before and after the program. C-reactive protein (CRP) in serum, matrix metalloproteinase (MMP)-8, MMP-9, and interleukin (IL)-1β in the gingival sulcus fluid were measured. After extracting bacterial genomic DNA from saliva, the presence of periodontal bacteria were detected using Taq probe. The relationship of each index before and after the program was analyzed through paired t-test and partial correlation analysis. Results: Campylobacter rectus (Cr) increased after the program, and there was no significant change in other bacteria. Serum CRP and Fusobacterium nucleatum (Fn), Aggregatibacter actinomycetemcomitans, Cr, ratio of Fn, and ratio of Cr had a positive relationship at baseline; however, the relationship was not significant after the program. Ratio of Prevotella intermedia had a positive relationship with MMP-9, MMP-8, IL-1β at baseline. Moreover, the ratio of Treponema denticola and the ratio of Tannerella forsythia showed a positive relationship with MMP-8, MMP-9, and IL-1β. The relationship between the ratio of Porphyromonas gingivalis and IL-1β showed a constant positive relationship at baseline and after the program. Conclusion: Obesity control program in subjects with a BMI of ≥23 kg/m² accompanied by diet and exercise did not affect the changes in periodontal bacteria itself, but changes in the relationship between periodontal bacteria and serum CRP, the relationship between the inflammatory index in the gingival crevicular fluid and periodontal bacteria was observed.

• 한국수산과학회지
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• 제54권6호
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• pp.927-937
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• 2021

This study was performed to screen the antimicrobial activities of the extract from the Pacific oyster Crassostrea gigas against skin pathogens and to purify the relevant antibacterial peptide. The acidified extract showed potent antibacterial activities against gram-positive and gram-negative bacteria but showed no activity against Candida albicans and no significant cell toxicity. Among acne-causing pathogens, the acidified extract showed potent antibacterial activity only against Staphylococcus aureus, and its antibacterial activity was completely abolished by treatment with trypsin or chymotrypsin, and was inhibited by salt treatment. The acidified extract showed strong DNA-binding ability but did not show bacterial membrane permeabilizing ability. Based on antimicrobial activity screening and cytotoxic effects, a novel antibacterial peptide was purified from the acidified gill extract using solid-phase extraction, cation-exchange, and reversed-phase HPLC. The resulting peptide had a molecular weight of 4800.8 Da and showed partial sequence homology with the carbonic anhydrase 4 (CA4) protein in the hard-shelled mussel. Overall, we purified a novel antibacterial peptide, named cgCAFLP, which is related to carbonic anhydrase 4 (CA4) protein, against skin pathogens. Our results suggest that the Pacific oyster extract could be used as an additive to control some acne-related skin pathogens (S. aureus).

• The Plant Pathology Journal
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• 제38권5호
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• pp.482-489
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• 2022

Fire blight caused by Erwinia amylovora (Ea) is a devastating disease in apple and pear trees. Oxolinic acid (OA), a quinolone family antibiotic that inhibits DNA gyrase, has been employed to control fire blight in South Korea since 2015. The continuous use of this bactericide has resulted in the emergence of OA-resistant strains in bacterial pathogens in other countries. To investigate the occurrence of OA-resistant Ea strains in South Korea, we collected a total of 516 Ea isolates from diseased apple and pear trees in 2020-2021 and assessed their sensitivities to OA. We found that all isolates were susceptible to OA. To explore the possibility of emerging OA-resistant Ea by continuous application of OA, we exposed Ea stains to a range of OA concentrations and constructed OA-resistant mutant strains. Resistance was associated with mutations in the GyrA at codons 81 and 83, which result in glycine to cysteine and serine to arginine amino acid substitutions, respectively. The in vitro growth of the mutants in nutrient media and their virulence in immature apple fruits were lower than those of wild-type. Our results suggest that OA-resistance decreases the fitness of Ea. Future work should clarify the mechanisms by which OA-resistance decreases virulence of this plant pathogen. Continuous monitoring of OA-resistance in Ea is required to maintain the efficacy of this potent bactericide.

• 한국수산과학회지
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• 제55권6호
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• pp.875-885
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• 2022

This study was performed to confirm the optimal extraction method for antimicrobial peptides from the Hard-shelled mussel. Extractions were performed with two processes including 1% HAc/boiling and 1% HAc/non-boiling methods and used extracts for the comparison of the antimicrobial activity, protease stability, action mechanism, AU-PAGE (acid-urea PAGE), and HPLC chromatograms. 1% HAc/boiling extract showed potent antibacterial activities both against Gram-positive and negative bacterium but 1% HAc/non-boiling extract showed antibacterial activity only against Gram-positive bacteria. Treatment of 1% HAc/boiling extract with proteases retained almost antibacterial activity against B. subtilis, but abolished significant antibacterial activity against E. coli D31. Only 1% HAc/boiling extract showed two discrete clearing antibacterial zones including slow migrating and rapid migrating zones. Both extracts showed strong DNA-binding ability but did not show bacterial membrane permeabilizing ability. In comparison of the chromatogram obtained from C₁₈ or cation-exchange HPLC, the eluted peaks from 1% HAc/boiling extract showed high hydrophobic property or absorbance compared to 1% HAc/non-boiling extract, respectively. The concentration of the purified antimicrobial peptide was also higher in 1% HAc/boiling extract than in 1% HAc/non-boiling extract. Our results suggest that the effective extraction condition for antimicrobial peptides from marine invertebrate is boiling process in a weak acetic acid solution (1%).

• 한국수산과학회지
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• 제56권1호
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• pp.99-108
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• 2023

In this study, changes in biofloc-related and pathogenic bacteria in both low and high concentration biofloc breeding water planted with the halophyte (Triglochin maritimum were investigated). In the low-concentration biofloc breeding water, the ratio of bacteria related to the nitrogen cycle was initially 59.57% and, it decreased to 26.57% at the end of the experiment while other bacteria (excluding nitrogen-cycling bacteria and vibrios) increased from 38.75% to 73.43%. However, the planted experimental group maintained a relatively high ratio of nitrogen cycling bacteria at 58%. In the high-concentration experimental group, bacteria related to the initial nitrogen cycle, non-pathogenic vibrios, and pathogenic vibrios were 11.60, 36.28, and 20.14%, respectively. Finally, nitrogen-cycling bacteria were 36.47% in the control group and 37.55% in the planted group. The total number of vibrios decreased by 46.54% in the planted group and 48.01% in the control group, indicating a significant decrease in both experimental groups. However, the residual rate of pathogenic vibrios was 4.48% in the control group and 0.54% in the planted group. Overall, the planted group showed decreasing harmful bacteria and increasing useful bacteria.