Journal of Microbiology

Journal Articles

Characterization and Comparative Genomic Analysis of vB_BceM_CEP1: A Novel Temperate Bacteriophage Infecting Burkholderia cepacia Complex.: Momen Askoura, Eslam K Fahmy, Safya E Esmaeel, Wael A H Hegazy, Aliaa Abdelghafar; J. Microbiol. 2024;62(11):1035-1055. Published online November 18, 2024; DOI: https://doi.org/10.1007/s12275-024-00185-2

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Abstract: The increasing prevalence of multidrug-resistant bacteria imminently threatens public health and jeopardizes nearly all aspects of modern medicine. The Burkholderia cepacia complex (Bcc) comprises Burkholderia cepacia and the related species of Gram-negative bacteria. Members of the Bcc group are opportunistic pathogens responsible for various chronic illnesses, including cystic fibrosis and chronic granulomatous disease. Phage therapy is emerging as a potential solution to combat the antimicrobial resistance crisis. In this study, a temperate phage vB_BceM_CEP1 was isolated from sewage and fully characterized. Transmission electron microscopy indicated that vB_BceM_CEP1 belongs to the family Peduoviridae. The isolated phage demonstrated enhanced environmental stability and antibiofilm potential. One-step growth analysis revealed a latent period of 30 min and an average burst size of 139 plaque-forming units per cell. The genome of vB_BceM_CEP1 consists of 32,486 bp with a GC content of 62.05%. A total of 40 open reading frames were annotated in the phage genome, and none of the predicted genes was annotated as tRNA. Notably, genes associated with antibiotic resistance, host virulence factors, and toxins were absent from the vB_BceM_CEP1 genome. Based on its unique phenotype and phylogeny, the isolated phage vB_BceM_CEP1 is classified as a new temperate phage with lytic activity. The findings of this study enhance our understanding of the diversity of Bcc phages.

Enterococcus Phage vB_EfaS_HEf13 as an Anti-Biofilm Agent Against Enterococcus faecalis.: Dongwook Lee, Jintaek Im, A Reum Kim, Woohyung Jun, Cheol-Heui Yun, Seung Hyun Han; J. Microbiol. 2024;62(8):683-693. Published online June 27, 2024; DOI: https://doi.org/10.1007/s12275-024-00150-z

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Abstract: Enterococcus faecalis is a Gram-positive bacterium that is frequently found in the periapical lesion of patients with apical periodontitis. Its biofilm formation in root canal is closely related to the development of refractory apical periodontitis by providing increased resistance to endodontic treatments. Phage therapy has recently been considered as an efficient therapeutic strategy in controlling various periodontal pathogens. We previously demonstrated the bactericidal capacities of Enterococcus phage vB_EfaS_HEf13 (phage HEf13) against clinically-isolated E. faecalis strains. Here, we investigated whether phage HEf13 affects biofilm formation and pre-formed biofilm of clinically-isolated E. faecalis, and its combinatory effect with endodontic treatments, including chlorhexidine (CHX) and penicillin. The phage HEf13 inhibited biofilm formation and disrupted pre-formed biofilms of E. faecalis in a dose- and time-dependent manner. Interestingly, phage HEf13 destroyed E. faecalis biofilm exopolysaccharide (EPS), which is known to be a major component of bacterial biofilm. Furthermore, combined treatment of phage HEf13 with CHX or penicillin more potently inhibited biofilm formation and disrupted pre-formed biofilm than either treatment alone. Confocal laser scanning microscopic examination demonstrated that these additive effects of the combination treatments on disruption of pre-formed biofilm are mediated by relatively enhanced reduction in thickness distribution and biomass of biofilm. Collectively, our results suggest that the effect of phage HEf13 on E. faecalis biofilm is mediated by its EPS-degrading property, and its combination with endodontic treatments more potently suppresses E. faecalis biofilm, implying that phage HEf13 has potential to be used as a combination therapy against E. faecalis infections.

[Protocol] Use of Cas9 Targeting and Red Recombination for Designer Phage Engineering: Shin-Yae Choi , Danitza Xiomara Romero-Calle , Han-Gyu Cho , Hee-Won Bae , You-Hee Cho; J. Microbiol. 2024;62(1):1-10. Published online February 1, 2024; DOI: https://doi.org/10.1007/s12275-024-00107-2

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Abstract: Bacteriophages (phages) are natural antibiotics and biological nanoparticles, whose application is significantly boosted by recent advances of synthetic biology tools. Designer phages are synthetic phages created by genome engineering in a way to increase the benefits or decrease the drawbacks of natural phages. Here we report the development of a straightforward genome engineering method to efficiently obtain engineered phages in a model bacterial pathogen, Pseudomonas aeruginosa. This was achieved by eliminating the wild type phages based on the Streptococcus pyogenes Cas9 (SpCas9) and facilitating the recombinant generation based on the Red recombination system of the coliphage λ (λRed). The producer (PD) cells of P. aeruginosa strain PAO1 was created by miniTn7-based chromosomal integration of the genes for SpCas9 and λRed under an inducible promoter. To validate the efficiency of the recombinant generation, we created the fluorescent phages from a temperate phage MP29. A plasmid bearing the single guide RNA (sgRNA) gene for selectively targeting the wild type gp35 gene and the editing template for tagging the Gp35 with superfolder green fluorescent protein (sfGFP) was introduced into the PD cells by electroporation. We found that the targeting efficiency was affected by the position and number of sgRNA. The fluorescent phage particles were efficiently recovered from the culture of the PD cells expressing dual sgRNA molecules. This protocol can be used to create designer phages in P. aeruginosa for both application and research purposes.

Comparative Transcriptomic Analysis of Flagellar‑Associated Genes in Salmonella Typhimurium and Its rnc Mutant: Seungmok Han , Ji-Won Byun , Minho Lee; J. Microbiol. 2024;62(1):33-48. Published online January 5, 2024; DOI: https://doi.org/10.1007/s12275-023-00099-5

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Abstract: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a globally recognized foodborne pathogen that affects both animals and humans. Endoribonucleases mediate RNA processing and degradation in the adaptation of bacteria to environmental changes and have been linked to the pathogenicity of S. Typhimurium. Not much is known about the specific regulatory mechanisms of these enzymes in S. Typhimurium, particularly in the context of environmental adaptation. Thus, this study carried out a comparative transcriptomic analysis of wild-type S. Typhimurium SL1344 and its mutant (Δrnc), which lacks the rnc gene encoding RNase III, thereby elucidating the detailed regulatory characteristics that can be attributed to the rnc gene. Global gene expression analysis revealed that the Δrnc strain exhibited 410 upregulated and 301 downregulated genes (fold-change > 1.5 and p < 0.05), as compared to the wild-type strain. Subsequent bioinformatics analysis indicated that these differentially expressed genes are involved in various physiological functions, in both the wild-type and Δrnc strains. This study provides evidence for the critical role of RNase III as a general positive regulator of flagellar-associated genes and its involvement in the pathogenicity of S. Typhimurium.

Characterization of antibiotic-resistant, coagulase-negative staphylococci from fresh produce and description of Staphylococcus shinii sp. nov. isolated from chives: Gyu-Sung Cho , Bo Li , Erik Brinks , Charles , M.A.P. Franz; J. Microbiol. 2022;60(9):877-889. Published online June 22, 2022; DOI: https://doi.org/10.1007/s12275-022-2100-5

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5 Citations

Abstract: Coagulase-negative Staphylococcus (CoNS) species may possess antibiotic resistance genes and have been associated with nosocomial infections. In this study, 91 CoNS with decreased susceptibility to oxacillin were isolated from fresh produce using oxacillin containing agar plates. Their antibiotic resistances were determined phenotypically and all isolates were identified by rep-PCR, 16S rRNA and rpoB gene sequencing. Furthermore, the genomes of representative strains were sequenced in order to confirm species identification by phylogenomics. The majority (64 of 91) of the CoNS strains could be identified as Mammaliicoccus (M.) fleurettii, while 13 were identified as M. sciuri, 8 as M. vitulinus, 2 as Staphylococcus (S.) epidermidis and single strains each as S. warneri, S. xylosus, Staphylococcus spp. and S. casei. Most of the strains were generally susceptible to clinically-relevant antibiotics, but only few (< 7%) strains possessed multiple resistances. Both oxacillin and cefoxitin resistant isolates were considered to be presumptive methicillin-resistant CoNS. From whole genome sequencing data of 6 representative strains, the mecA gene, accessory genes and the SCC loci were compared, which revealed high variability between some of the strains. The major fatty acids of K22-5MT strain included anteiso-C15:0, iso-C15:0, iso-C17:0, anteiso-C17:0, C18:0, and C20:0. Average nucleotide identity and digital DNA-DNA hybridization values indicated that Staphylococcus strain K22-5MT was below the species delineation cutoff values for ANI (less than 91%) and DDH (less than 44.4%), with the most closely related species being the S. pseudoxylosus S04009T type strain. Thus, strain K22- 5MT (=DSM 112532T, =LMG 32324T) represents a novel species, for which the name Staphylococcus shinii sp. nov. is proposed.

Eradication of drug-resistant Acinetobacter baumannii by cell-penetrating peptide fused endolysin: Jeonghyun Lim , Jaeyeon Jang , Heejoon Myung , Miryoung Song; J. Microbiol. 2022;60(8):859-866. Published online May 25, 2022; DOI: https://doi.org/10.1007/s12275-022-2107-y

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9 Citations

Abstract: Antimicrobial agents targeting peptidoglycan have shown successful results in eliminating bacteria with high selective toxicity. Bacteriophage encoded endolysin as an alternative antibiotics is a peptidoglycan degrading enzyme with a low rate of resistance. Here, the engineered endolysin was developed to defeat multiple drug-resistant (MDR) Acinetobacter baumannii. First, putative endolysin PA90 was predicted by genome analysis of isolated Pseudomonas phage PBPA. The His-tagged PA90 was purified from BL21(DE3) pLysS and tested for the enzymatic activity using Gram-negative pathogens known for having a high antibiotic resistance rate including A. baumannii. Since the measured activity of PA90 was low, probably due to the outer membrane, cell-penetrating peptide (CPP) DS4.3 was introduced at the N-terminus of PA90 to aid access to its substrate. This engineered endolysin, DS-PA90, completely killed A. baumannii at 0.25 μM, at which concentration PA90 could only eliminate less than one log in CFU/ml. Additionally, DS-PA90 has tolerance to NaCl, where the ~50% of activity could be maintained in the presence of 150 mM NaCl, and stable activity was also observed with changes in pH or temperature. Even MDR A. baumannii strains were highly susceptible to DS-PA90 treatment: five out of nine strains were entirely killed and four strains were reduced by 3–4 log in CFU/ml. Consequently, DS-PA90 could protect waxworm from A. baumannii-induced death by ~70% for ATCC 17978 or ~44% for MDR strain 1656-2 infection. Collectively, our data suggest that CPP-fused endolysin can be an effective antibacterial agent against Gramnegative pathogens regardless of antibiotics resistance mechanisms.

Potent antibacterial and antibiofilm activities of TICbf-14, a peptide with increased stability against trypsin: Liping Wang , Xiaoyun Liu , Xinyue Ye , Chenyu Zhou , Wenxuan Zhao , Changlin Zhou , Lingman Ma; J. Microbiol. 2022;60(1):89-99. Published online December 29, 2021; DOI: https://doi.org/10.1007/s12275-022-1368-9

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Abstract: The poor stability of peptides against trypsin largely limits their development as potential antibacterial agents. Here, to obtain a peptide with increased trypsin stability and potent antibacterial activity, TICbf-14 derived from the cationic peptide Cbf-14 was designed by the addition of disulfide-bridged hendecapeptide (CWTKSIPPKPC) loop. Subsequently, the trypsin stability and antimicrobial and antibiofilm activities of this peptide were evaluated. The possible mechanisms underlying its mode of action were also clarified. The results showed that TICbf-14 exhibited elevated trypsin inhibitory activity and effectively mitigated lung histopathological damage in bacteria-infected mice by reducing the bacterial counts, further inhibiting the systemic dissemination of bacteria and host inflammation. Additionally, TICbf-14 significantly repressed bacterial swimming motility and notably inhibited biofilm formation. Considering the mode of action, we observed that TICbf-14 exhibited a potent membrane-disruptive mechanism, which was attributable to its destructive effect on ionic bridges between divalent cations and LPS of the bacterial membrane. Overall, TICbf-14, a bifunctional peptide with both antimicrobial and trypsin inhibitory activity, is highly likely to become an ideal candidate for drug development against bacteria.

Interaction between hypoviral-regulated fungal virulence factor laccase3 and small heat shock protein Hsp24 from the chestnut blight fungus Cryphonectria parasitica: Jeesun Chun† , Yo-Han Ko† , Dae-Hyuk Kim; J. Microbiol. 2022;60(1):57-62. Published online November 26, 2021; DOI: https://doi.org/10.1007/s12275-022-1498-0

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Abstract: Laccase3 is an important virulence factor of the fungus Cryphonectria parasitica. Laccase3 gene (lac3) transcription is induced by tannic acid, a group of phenolic compounds found in chestnut trees, and its induction is regulated by the hypovirus CHV1 infection. CpHsp24, a small heat shock protein gene of C. parasitica, plays a determinative role in stress adaptation and pathogen virulence. Having uncovered in our previous study that transcriptional regulation of the CpHsp24 gene in response to tannic acid supplementation and CHV1 infection was similar to that of the lac3, and that conserved phenotypic changes of reduced virulence were observed in mutants of both genes, we inferred that both genes were implicated in a common pathway. Building on this finding, in this paper we examined whether the CpHsp24 protein (CpHSP24) was a molecular chaperone for the lac3 protein (LAC3). Our pull-down experiment indicated that the protein products of the two genes directly interacted with each other. Heterologous co-expression of CpHsp24 and lac3 genes using Saccharomyces cerevisiae resulted in more laccase activity in the cotransformant than in a parental lac3-expresssing yeast strain. These findings suggest that CpHSP24 is, in fact, a molecular chaperone for the LAC3, which is critical component of fungal pathogenesis.

Raman spectroscopy reveals alteration of spore compositions under different nutritional conditions in Lysinibacillus boronitolerans YS11: Youngung Ryu , Minyoung Hong , Soo Bin Kim , Tae Kwon Lee , Woojun Park; J. Microbiol. 2021;59(5):491-499. Published online March 29, 2021; DOI: https://doi.org/10.1007/s12275-021-0679-6

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6 Citations

Abstract: Little is known about final spores components when bacteria undergo sporulation under different nutrient conditions. Different degrees of resistance and germination rates were observed in the three types of spores of Lysinibacillus boronitolerans YS11 (SD, Spores formed in Difco sporulation mediumTM; SC and SF, Spores formed in an agricultural byproduct medium with 10 mM CaCl2 and with 10 mM FeSO4, respectively). Stronger UV resistance was recorded for SF with 1.8–2.3-fold greater survival than SC and SD under UV treatment. The three spore types showed similar heat resistances at 80°C, but survival rates of SC and SD were much higher (~1,000 times) than those of SF at 90°C. However, germination capacity of SF was 20% higher than those of SD and SC on Luria-Bertani agar plates for 24 h. SF germinated more rapidly in a liquid medium with high NaCl concentrations than SC and SD, but became slower under alkaline conditions. Raman spectroscopy was used to analyze the heterogeneities in the three types of vegetative cells and their spores under different nutritional conditions. Exponentially grown-each vegetative cells had different overall Raman peak values. Raman peaks of SC, SD, and SF also showed differences in adenine and amide III compositions and nucleic acid contents. Our data along with Raman spectroscopy provided the evidence that spores formed under under different growth conditions possess very different cellular components, which affected their survival and germination rates.

Molecular mechanism of Escherichia coli H10407 induced diarrhoea and its control through immunomodulatory action of bioactives from Simarouba amara (Aubl.): Hegde Veena , Sandesh K. Gowda , Rajeshwara N. Achur , Nayaka Boramuthi Thippeswamy; J. Microbiol. 2021;59(4):435-447. Published online February 25, 2021; DOI: https://doi.org/10.1007/s12275-021-0423-2

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Abstract: Enterotoxigenic Escherichia coli (ETEC) infection is a major cause of death in children under the age of five in developing countries. ETEC (O78:H11:CFA/I:LT+:ST+) mechanism has been studied in detail with either heat labile (LT) or heat stable (ST) toxins using in vitro and in vivo models. However, there is no adequate information on ETEC pathogenesis producing both the toxins (LT, ST) in BALB/c mice model. In this study, female mice have been employed to understand ETEC H10407 infection induced changes in physiology, biochemical and immunological patterns up to seven days post-infection and the antidiarrhoeal effect of Simarouba amara (Aubl.) bark aqueous extract (SAAE) has also been looked into. The results indicate that BALB/c is sensitive to ETEC infection resulting in altered jejunum and ileum histomorphology. Withal, ETEC influenced cAMP, PGE2, and NO production resulting in fluid accumulation with varied Na+, K+, Cl-, and Ca2+ levels. Meanwhile, ETEC subverted expression of IL-1β, intestine alkaline phosphatase (IAP), and myeloperoxidase (MPO) in jejunum and ileum. Our data also indicate the severity of pathogenesis reduction which might be due to attainment of equilibrium after reaching optimum rate of infection. Nevertheless, degree of pathogenesis was highly significant (p < 0.01) in all the studied parameters. Besides that, SAAE was successful in reducing the infectious diarrhoea by inhibiting ETEC H10407 in intestine (jejunum and ileum), and shedding in feces. SAAE decreased cAMP, PGE2, and fluid accumulation effectively and boosted the functional activity of immune system in jejunum and ileum IAP, MPO, IL-1β, and nitric oxide.

Review

Dissection of plant microbiota and plant-microbiome interactions: Kihyuck Choi , Raees Khan , Seon-Woo Lee; J. Microbiol. 2021;59(3):281-291. Published online February 23, 2021; DOI: https://doi.org/10.1007/s12275-021-0619-5

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36 Citations

Abstract: Plants rooted in soil have intimate associations with a diverse array of soil microorganisms. While the microbial diversity of soil is enormous, the predominant bacterial phyla associated with plants include Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia. Plants supply nutrient niches for microbes, and microbes support plant functions such as plant growth, development, and stress tolerance. The interdependent interaction between the host plant and its microbes sculpts the plant microbiota. Plant and microbiome interactions are a good model system for understanding the traits in eukaryotic organisms from a holobiont perspective. The holobiont concept of plants, as a consequence of co-evolution of plant host and microbiota, treats plants as a discrete ecological unit assembled with their microbiota. Dissection of plant-microbiome interactions is highly complicated; however, some reductionist approaches are useful, such as the synthetic community method in a gnotobiotic system. Deciphering the interactions between plant and microbiome by this reductionist approach could lead to better elucidation of the functions of microbiota in plants. In addition, analysis of microbial communities’ interactions would further enhance our understanding of coordinated plant microbiota functions. Ultimately, better understanding of plantmicrobiome interactions could be translated to improvements in plant productivity.

Journal Articles

Leucobacter coleopterorum sp. nov., Leucobacter insecticola sp. nov., and Leucobacter viscericola sp. nov., isolated from the intestine of the diving beetles, Cybister brevis and Cybister lewisianus, and emended description of the genus Leucobacter: Dong-Wook Hyun , Hojun Sung , Pil Soo Kim , Ji-Hyun Yun , Jin-Woo Bae; J. Microbiol. 2021;59(4):360-368. Published online January 26, 2021; DOI: https://doi.org/10.1007/s12275-021-0472-6

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Abstract: Three novel bacterial strains, HDW9AT, HDW9BT, and HDW9CT, isolated from the intestine of the diving beetles Cybister lewisianus and Cybister brevis, were characterized as three novel species using a polyphasic approach. The isolates were Gram-staining-positive, strictly aerobic, non-motile, and rod-shaped. They grew optimally at 30°C (pH 7) in the presence of 0.5% (wt/vol) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that they belong to the genus Leucobacter and are closely related to L. denitrificans M1T8B10T (98.4–98.7% sequence similarity). Average nucleotide identity (ANI) values among the isolates were 76.4–84.1%. ANI values for the isolates and the closest taxonomic species, L. denitrificans KACC 14055T, were 72.3–73.1%. The isolates showed ANI values of < 76.5% with all analyzable Leucobacter strains in the EzBioCloud database. The genomic DNA G + C content of the isolates was 60.3–62.5%. The polar lipid components were phosphatidylglycerol, diphosphatidylglycerol, and other unidentified glycolipids, phospholipids, and lipids. The major cellular fatty acids were anteiso- C15:0, iso-C16:0, and anteiso-C17:0. MK-10 was the major respiratory quinone, and MK-7 and MK-11 were the minor respiratory quinones. The whole-cell sugar components of the isolates were ribose, glucose, galactose, and mannose. The isolates harbored L-2,4-diaminobutyric acid, L-serine, L-lysine, L-aspartic acid, glycine, and D-glutamic acid within the cell wall peptidoglycan. Based on phylogenetic, phenotypic, chemotaxonomic, and genotypic analyses, strains HDW9AT, HDW9BT, and HDW9CT represent three novel species within the genus Leucobacter. We propose the name Leucobacter coleopterorum sp. nov. for strain HDW9AT (= KACC 21331T = KCTC 49317T = JCM 33667T), the name Leucobacter insecticola sp. nov. for strain HDW9BT (= KACC 21332T = KCTC 49318T = JCM 33668T), and the name Leucobacter viscericola sp. nov. for strain HDW9CT (= KACC 21333T = KCTC 49319T = JCM 33669T).

Soil water content as a critical factor for stable bacterial community structure and degradative activity in maritime Antarctic soil: Dockyu Kim , Namyi Chae , Mincheol Kim , Sungjin Nam , Eungbin Kim , Hyoungseok Lee; J. Microbiol. 2020;58(12):1010-1017. Published online December 2, 2020; DOI: https://doi.org/10.1007/s12275-020-0490-9

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Abstract: Recent increases in air temperature across the Antarctic Peninsula may prolong the thawing period and directly affect the soil temperature (Ts) and volumetric soil water content (SWC) in maritime tundra. Under an 8°C soil warming scenario, two customized microcosm systems with maritime Antarctic soils were incubated to investigate the differential influence of SWC on the bacterial community and degradation activity of humic substances (HS), the largest constituent of soil organic carbon and a key component of the terrestrial ecosystem. When the microcosm soil (KS1-4Feb) was incubated for 90 days (T = 90) at a constant SWC of ~32%, the initial HS content (167.0 mg/g of dried soil) decreased to 156.0 mg (approximately 6.6% loss, p < 0.05). However, when another microcosm soil (KS1-4Apr) was incubated with SWCs that gradually decreased from 37% to 9% for T = 90, HS degradation was undetected. The low HS degradative activity persisted, even after the SWC was restored to 30% with water supply for an additional T = 30. Overall bacterial community structure remained relatively stable at a constant SWC setting (KS1-4Feb). In contrast, we saw marked shifts in the bacterial community structure with the changing SWC regimen (KS1-4Apr), suggesting that the soil bacterial communities are vulnerable to drying and re-wetting conditions. These microcosm experiments provide new information regarding the effects of constant SWC and higher Ts on bacterial communities for HS degradation in maritime Antarctic tundra soil.

Construction of a genetically modified T7Select phage system to express the antimicrobial peptide 1018: David J. Lemon , Matthew K. Kay , James K. Titus , April A. Ford , Wen Chen , LCDR Nicholas J. Hamlin , Yoon Y. Hwang; J. Microbiol. 2019;57(6):532-538. Published online May 27, 2019; DOI: https://doi.org/10.1007/s12275-019-8686-6

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23 Citations

Abstract: Bacteriophage therapy was an ascendant technology for combating bacterial infections before the golden age of antibiotics, but the therapeutic potential of phages was largely ignored after the discovery of penicillin. Recently, with antibioticresistant infections on the rise, these phages are receiving renewed attention to combat problematic bacterial infections. Our approach is to enhance bacteriophages with antimicrobial peptides, short peptides with broad-spectrum antibiotic or antibiofilm effects. We inserted coding sequences for 1018, an antimicrobial peptide previously shown to be an effective broad-spectrum antimicrobial and antibiofilm agent, or the fluorescent marker mCherry, into the T7Select phage genome. Transcription and production of 1018 or mCherry began rapidly after E. coli cultures were infected with genetically modified phages. mCherry fluorescence, which requires a 90 min initial maturation period, was observed in infected cultures after 2 h of infection. Finally, we tested phages expressing 1018 (1018 T7) against bacterial planktonic cultures and biofilms, and found the 1018 T7 phage was more effective than the unmodified T7Select phage at both killing planktonic cells and eradicating established biofilms, validating our phage-driven antimicrobial peptide expression system. The combination of narrow-spectrum phages delivering relatively high local doses of broad-spectrum antimicrobials could be a powerful
method
to combat resistant infections. The experiments we describe prove this combination is feasible in vitro, but further testing and optimization are required before genetically modified phages are ready for use in vivo.

PROTOCOL] Applications of different solvents and conditions for differential extraction of lipopolysaccharide in Gram-negative bacteria: Mai Phuong Nguyen , Le Viet Ha Tran , Hyun Namgoong , Yong-Hak Kim; J. Microbiol. 2019;57(8):644-654. Published online May 23, 2019; DOI: https://doi.org/10.1007/s12275-019-9116-5

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Abstract: Lipopolysaccharide (LPS) is one of the major components in the outer membrane of Gram-negative bacteria. However, its heterogeneity and variability in different bacteria and differentiation conditions make it difficult to extract all of the structural variants. We designed a solution to improve quality and biological activity of LPS extracted from various bacteria with different types of LPS, as compared to conventional
methods
. We introduced a quality index as a simple measure of LPS purity in terms of a degree of polysaccharide content detected by absorbance at 204 nm. Further experiments using gel electrophoresis, endotoxin test, and macrophage activation test were performed to evaluate the performance and reliability of a proposed ‘T-sol’ method and the biological effectiveness and character of the LPS products. We presented that the T-sol method had differential effects on extraction of a RAW 264.7 cell-activating LPS, which was effective in the macrophage activation with similar effects in stimulating the production of TNF-alpha. In conclusion, the T-sol method provides a simple way to improve quality and biological activity of LPS with high yield.

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