Journal of Microbiology

Journal Articles

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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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.

Transposon insertion site sequencing (TIS) of Pseudomonas aeruginosa: Hongbaek Cho; J. Microbiol. 2021;59(12):1067-1074. Published online December 4, 2021; DOI: https://doi.org/10.1007/s12275-021-1565-y

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Abstract: Transposon insertion site sequencing (TIS) is a technique that determines the insertion profile of a transposon mutant library by massive parallel sequencing of transposon-genomic DNA junctions. Because the transposon insertion profile reflects the abundance of each mutant in the library, it provides information to assess the fitness contribution of each genetic locus of a bacterial genome in a specific growth condition or strain background. Although introduced only about a dozen years ago, TIS has become an important tool in bacterial genetics that provides clues to study biological functions and regulatory mechanisms. Here, I describe a protocol for generating high density transposon insertion mutant libraries and preparing Illumina sequencing samples for mapping the transposon junctions of the transposon mutant libraries using Pseudomonas aeruginosa as an example.

Development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) thermal inactivation method with preservation of diagnostic sensitivity: Young-Il Kim , Mark Anthony B. Casel , Se-Mi Kim , Seong-Gyu Kim , Su-Jin Park , Eun-Ha Kim , Hye Won Jeong , Haryoung Poo , Young Ki Choi; J. Microbiol. 2020;58(10):886-891. Published online September 29, 2020; DOI: https://doi.org/10.1007/s12275-020-0335-6

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Abstract: Various treatments and agents had been reported to inactivate RNA viruses. Of these, thermal inactivation is generally considered an effective and cheap method of sample preparation for downstream assays. The purpose of this study is to establish a safe inactivation method for SARS-CoV-2 without compromising the amount of amplifiable viral genome necessary for clinical diagnoses. In this study, we demonstrate the infectivity and genomic stability of SARSCoV- 2 by thermal inactivation at both 56°C and 65°C. The
results
substantiate that viable SARS-CoV-2 is readily inactivated when incubated at 56°C for 30 min or at 65°C for 10 min. qRT-PCR of specimens heat-inactivated at 56°C for 30 min or 65°C for 15 min revealed similar genomic RNA stability compared with non-heat inactivated specimens. Further, we demonstrate that 30 min of thermal inactivation at 56°C could inactivate viable viruses from clinical COVID-19 specimens without attenuating the qRT-PCR diagnostic sensitivity. Heat treatment of clinical specimens from COVID-19 patients at 56°C for 30 min or 65°C for 15 min could be a useful
method
for the inactivation of a highly contagious agent, SARS-CoV-2. Use of this method would reduce the potential for secondary infections in BSL2 conditions during diagnostic procedures. Importantly, infectious virus can be inactivated in clinical specimens without compromising the sensitivity of the diagnostic RT-PCR assay.

Georgenia faecalis sp. nov. isolated from the faeces of Tibetan antelope: Xiaoxia Wang , Jing Yang , Yuyuan Huang , Xiaomin Wu , Licheng Wang , Limei Han , Sha Li , Huan Li , Xiaoying Fu , Hai Chen , Xiong Zhu; J. Microbiol. 2020;58(9):734-740. Published online July 24, 2020; DOI: https://doi.org/10.1007/s12275-020-0060-1

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Abstract: Two aerobic, Gram-stain-positive, non-motile, non-sporulating coccoid strains, designated ZLJ0423T and ZLJ0321, were isolated from the faeces of Tibetan antelope (Pantholops hodgsonii). Their optimal temperature, NaCl concentration and pH for growth were 28°C, 0.5% (w/v) NaCl and pH 7.5, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains ZLJ0423T and ZLJ0321 were very similar to each other (99.8%) and had a sequence similarity of 97.0% with Georgenia satyanarayanai NBRC 107612T and Georgenia subflava CGMCC 1.12782T. Phylogenomic analysis based on 688 core genes indicated that these strains formed a clade with G. satyanarayanai NBRC 107612T and Georgenia wutianyii Z294T. The predominant cellular fatty acids were anteiso-C15:0, anteiso-C15:1 A and C16:0. The major menaquinone was MK-8(H4). The cell-wall amino acids consisted of alanine, lysine, glycine and aspartic acid, with lysine as the diagnostic diamino acid. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides and two unidentified lipids formed the polar lipid profile. The DNA G + C content of both isolates was 73.9 mol%. The digital DNA–DNA hybridization value between strains ZLJ0423T and ZLJ0321 was 91.2%, but their values with closely related species and other available type strains of the genus Georgenia were lower than the 70% threshold. On the basis of polyphasic taxonomic data, strains ZLJ0423T and ZLJ0321 represent a novel species within the genus Georgenia, for which the name Georgenia faecalis sp. nov. is proposed. The type strain is ZLJ0423T (= CGMCC 1.13681T = JCM 33470T).

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