Journal of Microbiology

Volume 59(6); June 2021

Review

[MINIREVIEW]Gain and loss of antibiotic resistant genes in multidrug resistant bacteria: One Health perspective: Misung Kim , Jaeeun Park , Mingyeong Kang , Jihye Yang , Woojun Park; J. Microbiol. 2021;59(6):535-545. Published online April 20, 2021; DOI: https://doi.org/10.1007/s12275-021-1085-9

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Abstract: The emergence of multidrug resistance (MDR) has become a global health threat due to the increasing unnecessary use of antibiotics. Multidrug resistant bacteria occur mainly by accumulating resistance genes on mobile genetic elements (MGEs), made possible by horizontal gene transfer (HGT). Humans and animal guts along with natural and engineered environments such as wastewater treatment plants and manured soils have proven to be the major reservoirs and hotspots of spreading antibiotic resistance genes (ARGs). As those environments support the dissemination of MGEs through the complex interactions that take place at the human-animalenvironment interfaces, a growing One Health challenge is for multiple sectors to communicate and work together to prevent the emergence and spread of MDR bacteria. However, maintenance of ARGs in a bacterial chromosome and/or plasmids in the environments might place energy burdens on bacterial fitness in the absence of antibiotics, and those unnecessary ARGs could eventually be lost. This review highlights and summarizes the current investigations into the gain and loss of ARG genes in MDR bacteria among human-animal- environment interfaces. We also suggest alternative treatments such as combinatory therapies or sequential use of different classes of antibiotics/adjuvants, treatment with enzymeinhibitors, and phage therapy with antibiotics to solve the MDR problem from the perspective of One Health issues.

Journal Articles

Pikeienuella piscinae gen. nov., sp. nov., a novel genus in the family Rhodobacteraceae: Jeeeun Park , Young-Sam Kim , Seong-Jin Kim , Sang-Eon Kim , Hyun-Kyoung Jung , Min-Ju Yu , Young Jae Jeon , Kyoung-Ho Kim; J. Microbiol. 2021;59(6):546-551. Published online April 20, 2021; DOI: https://doi.org/10.1007/s12275-021-0678-7

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Abstract: A novel bacterium, designated strain RR4-56T, was isolated from a biofilter of a seawater recirculating aquaculture system. The 16S rRNA gene sequence analysis showed that the isolate was closely related to Halovulum dunhuangense YYQ- 30T (92.6%), Albimonas donghaensis DS2T (91.3%), Pontivivens insulae GYSW-23T (91.3%), and Monaibacterium marinum C7T (90.9%), belonging to the family Rhodobacteraceae. The strain was aerobic, Gram-negative, rod-shaped, oxidasepositive, and catalase-negative. Its optimum temperature, pH, and salinity for growth were 25–30°C, pH 8.5, and 2–3% NaCl (w/v), respectively. Its growth occurred at 15–35°C, pH 5.0–9.5, and 0–7% NaCl (w/v). It contained ubiquinone-10 (Q-10), a respiratory quinone, and the major cellular fatty acids were 11-methyl C18:1 ω7c (31.9%), C18:1 ω6c (30.4%), and C19:0 cyclo ω8c (16.1%). The polar lipids present in the strain were phosphatidylglycerol, an unidentified phospholipid, and an unidentified aminolipid. The strain had one 4,373,045 bp circular chromosome with G + C contents of 65.9 mol% including 4,169 genes, 4,118 coding sequences (CDSs), 3 rRNAs, and 45 tRNAs. Genome annotation predicted some gene clusters related to the degradation of several types of organic matter such as protocatechuate, catechol, and phthalate. Based on the polyphasic characteristics, RR4-56T represents a novel genus and species in the family Rhodobacteraceae, for which the name Pikeienuella piscinae gen. nov., sp. nov. was proposed. The type strain is RR4-56T (= KCTC 52648T = DSM 107918T).

Description of Nocardioides piscis sp. nov., Sphingomonas piscis sp. nov. and Sphingomonas sinipercae sp. nov., isolated from the intestine of fish species Odontobutis interrupta (Korean spotted sleeper) and Siniperca scherzeri (leopard mandarin fish): Dong-Wook Hyun , Yun-Seok Jeong , Jae-Yun Lee , Hojun Sung , So-Yeon Lee , Jee-Won Choi , Hyun Sik Kim , Pil Soo Kim , Jin-Woo Bae; J. Microbiol. 2021;59(6):552-562. Published online April 20, 2021; DOI: https://doi.org/10.1007/s12275-021-1036-5

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Abstract: A polyphasic taxonomic approach was used to characterize three novel bacterial strains, designated as HDW12AT, HDW- 15BT, and HDW15CT, isolated from the intestine of fish species Odontobutis interrupta or Siniperca scherzeri. All isolates were obligate aerobic, non-motile bacteria, and grew optimally at 30°C. Phylogenetic analysis based on 16S rRNA sequences revealed that strain HDW12AT was a member of the genus Nocardioides, and closely related to Nocardioides allogilvus CFH 30205T (98.9% sequence identities). Furthermore, strains HDW15BT and HDW15CT were members of the genus Sphingomonas, and closely related to Sphingomonas lutea JS5T and Sphingomonas sediminicola Dae 20T (97.1% and 97.9% sequence identities), respectively. Strain HDW12AT contained MK-8 (H4), and strains HDW15BT and HDW15CT contained Q-10 as the respiratory quinone. Major polar lipid components of strain HDW12AT were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylinositol, and those of strains HDW15BT and HDW15CT were sphingoglycolipid, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine. The G + C content of strains HDW12AT, HDW15BT, and HDW15CT were 69.7, 63.3, and 65.5%, respectively. The results of phylogenetic, phenotypic, chemotaxonomic, and genotypic analyses suggest that strain HDW12AT represents a novel species within the genus Nocardioides, and strains HDW15BT and HDW15CT represent two novel species within the genus Sphingomonas. We propose the names Nocardioides piscis for strain HDW12AT (= KACC 21336T = KCTC 49321T = JCM 33670T), Sphingomonas piscis for strain HDW15BT (= KACC 21341T = KCTC 72588T = JCM 33738T), and Sphingomonas sinipercae for strain HDW15CT (= KACC 21342T = KCTC 72589T = JCM 33739T).

Deep convolutional neural network: a novel approach for the detection of Aspergillus fungi via stereomicroscopy: Haozhong Ma , Jinshan Yang , Xiaolu Chen , Xinyu Jiang , Yimin Su , Shanlei Qiao , Guowei Zhong; J. Microbiol. 2021;59(6):563-572. Published online March 29, 2021; DOI: https://doi.org/10.1007/s12275-021-1013-z

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Abstract: Fungi of the genus Aspergillus are ubiquitously distributed in nature, and some cause invasive aspergillosis (IA) infections in immunosuppressed individuals and contamination in agricultural products. Because microscopic observation and molecular detection of Aspergillus species represent the most operator-dependent and time-intensive activities, automated and cost-effective approaches are needed. To address this challenge, a deep convolutional neural network (CNN) was used to investigate the ability to classify various Aspergillus species. Using a dissecting microscopy (DM)/stereomicroscopy platform, colonies on plates were scanned with a 35× objective, generating images of sufficient resolution for classification. A total of 8,995 original colony images from seven Aspergillus species cultured in enrichment medium were gathered and autocut to generate 17,142 image crops as training and test datasets containing the typical representative morphology of conidiophores or colonies of each strain. Encouragingly, the Xception model exhibited a classification accuracy of 99.8% on the training image set. After training, our CNN model achieved a classification accuracy of 99.7% on the test image set. Based on the Xception performance during training and testing, this classification algorithm was further applied to recognize and validate a new set of raw images of these strains, showing a detection accuracy of 98.2%. Thus, our study demonstrated a novel concept for an artificial-intelligence-based and cost-effective detection
method
ology for Aspergillus organisms, which also has the potential to improve the public’s understanding of the fungal kingdom.

Comparative genomics analysis of Pediococcus acidilactici species: Zhenzhen Li , Qi Song , Mingming Wang , Junli Ren , Songling Liu , Shancen Zhao; J. Microbiol. 2021;59(6):573-583. Published online May 15, 2021; DOI: https://doi.org/10.1007/s12275-021-0618-6

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Abstract: Pediococcus acidilactici is a reliable bacteriocin producer and a promising probiotic species with wide application in the food and health industry. However, the underlying genetic features of this species have not been analyzed. In this study, we performed a comprehensive comparative genomic analysis of 41 P. acidilactici strains from various ecological niches. The bacteriocin production of 41 strains were predicted and three kinds of bacteriocin encoding genes were identified in 11 P. acidilactici strains, namely pediocin PA-1, enterolysin A, and colicin-B. Moreover, whole-genome analysis showed a high genetic diversity within the population, mainly related to a large proportion of variable genomes, mobile elements, and hypothetical genes obtained through horizontal gene transfer. In addition, comparative genomics also facilitated the genetic explanation of the adaptation for host environment, which specify the protection mechanism against the invasion of foreign DNA (i.e. CRISPR/Cas locus), as well as carbohydrate fermentation. The 41 strains of P. acidilactici can metabolize a variety of carbon sources, which enhances the adaptability of this species and survival in different environments. This study evaluated the antibacterial ability, genome evolution, and ecological flexibility of P. acidilactici from the perspective of genetics and provides strong supporting evidence for its industrial development and application.

Crystal structure of the nuclease and capping domain of SbcD from Staphylococcus aureus: Jinwook Lee , Inseong Jo , Jinsook Ahn , Seokho Hong , Soyeon Jeong , Aeran Kwon , Nam-Chul Ha; J. Microbiol. 2021;59(6):584-589. Published online April 20, 2021; DOI: https://doi.org/10.1007/s12275-021-1012-0

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Abstract: The SbcCD complex is an essential component of the DNA double-strand break (DSB) repair system in bacteria. The bacterial SbcCD complex recognizes and cleaves the DNA ends in DSBs by ATP-dependent endo- and exonuclease activities as an early step of the DNA repair process. SbcD consists of nuclease, capping, and helix-loop-helix domains. Here, we present the crystal structure of a SbcD fragment from Staphylococcus aureus, which contained nuclease and capping domains, at a resolution of 2.9 Å. This structure shows a dimeric assembly similar to that of the corresponding domains of SbcD from Escherichia coli. The S. aureus SbcD fragment exhibited endonuclease activities on supercoiled DNA and exonuclease activity on linear and nicked DNA. This study contributes to the understanding of the molecular basis for how bacteria can resist sterilizing treatment, causing DNA damage.

Detection of colistin-resistant populations prior to antibiotic exposure in KPC-2-producing Klebsiella pneumoniae clinical isolates: Jungyu Seo , Yu Mi Wi , Jong Min Kim , Yae-Jean Kim , Kwan Soo Ko; J. Microbiol. 2021;59(6):590-597. Published online March 29, 2021; DOI: https://doi.org/10.1007/s12275-021-0610-1

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Abstract: Although colistin is frequently regarded as the antibiotic of last resort in treating carbapenem-resistant Klebsiella pneumoniae, colistin heteroresistance may in part be associated with antibiotic treatment failure. However, we do not know how widespread the colistin heteroresistance is in carbapenem- resistant K. pneumoniae isolates. In this study, we performed colistin disc diffusion assays, E-tests, and population analysis profiling for KPC-2-producing K. pneumoniae isolates to identify colistin heteroresistance. Although no colistin- resistant colonies were detected by the disc diffusion test and E-test, a colistin-resistant subpopulation was identified in population analysis profiling in all colistin-susceptible, KPC-2-producing K. pneumoniae isolates. Colistin-resistant subpopulations were also identified even when isolates had no colistin exposure. The ratio of colistin-resistant subpopulations to the total population increased as the exposure concentration of colistin increased. In in vitro time-kill assays, regrowth was observed in all isolates after 2 h upon exposure to colistin. We identified common amino acid alterations in PhoQ, PhoP, and PmrB in colistin-resistant subpopulations from some isolates, but no substitutions were found in most resistant subpopulations from other isolates. In all colistin-resistant subpopulations, overexpression of PhoQ and PbgP was observed. In this study, we demonstrated that colistin heteroresistance may be common in KPC-2-producing K. pneumoniae isolates, which could not be detected in the disc diffusion method and E-test. Colistin heteroresistance may cause colistin treatment failure in part and may evolve into resistance. Thus, development of more reliable diagnostic methods is required to detect colistin heteroresistance.

Molecular characterization of the Saccharomycopsis fibuligera ATF genes, encoding alcohol acetyltransferase for volatile acetate ester formation: Hye Yun Moon , Hyeon Jin Kim , Ki Seung Kim , Su Jin Yoo , Dong Wook Lee , Hee Je Shin , Jeong Ah Seo , Hyun Ah Kang; J. Microbiol. 2021;59(6):598-608. Published online May 29, 2021; DOI: https://doi.org/10.1007/s12275-021-1159-8

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Abstract: Aroma ester components produced by fermenting yeast cells via alcohol acetyltransferase (AATase)-catalyzed intracellular reactions are responsible for the fruity character of fermented alcoholic beverages, such as beer and wine. Acetate esters are reportedly produced at relatively high concentrations by non-Saccharomyces species. Here, we identified 12 ATF orthologues (SfATFs) encoding putative AATases, in the diploid genome of Saccharomycopsis fibuligera KJJ81, an isolate from wheat-based Nuruk in Korea. The identified SfATF proteins (SfAtfp) display low sequence identities with S. cerevisiae Atf1p (between 13.3 and 27.0%). All SfAtfp identified, except SfAtf(A)4p and SfAtf(B)4p, contained the activation domain (HXXXD) conserved in other Atf proteins. Culture supernatant analysis using headspace gas chromatography mass spectrometry confirmed that the recombinant S. cerevisiae strains expressing SfAtf(A)2p, SfAtf(B)2p, and SfAtf(B)6p produced high levels of isoamyl and phenethyl acetates. The volatile aroma profiles generated by the SfAtf proteins were distinctive from that of S. cerevisiae Atf1p, implying difference in the substrate preference. Cellular localization analysis using GFP fusion revealed the localization of SfAtf proteins proximal to the lipid particles, consistent with the presence of amphipathic helices at their N- and C-termini. This is the first report that systematically characterizes the S. fibuligera ATF genes encoding functional AATases responsible for acetate ester formation using higher alcohols as substrate, demonstrating their biotechnological potential for volatile ester production.

UBCG2: Up-to-date bacterial core genes and pipeline for phylogenomic analysis: Jihyeon Kim , Seong-In Na , Dongwook Kim , Jongsik Chun; J. Microbiol. 2021;59(6):609-615. Published online May 29, 2021; DOI: https://doi.org/10.1007/s12275-021-1231-4

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106 Citations

Abstract: Phylogenomic tree reconstruction has recently become a routine and critical task to elucidate the evolutionary relationships among bacterial species. The most widely used method utilizes the concatenated core genes, universally present in a single-copy throughout the bacterial domain. In our previous study, a bioinformatics pipeline termed Up-to-date Bacterial Core Genes (UBCG) was developed with a set of bacterial core genes selected from 1,429 species covering 28 phyla. In this study, we revised a new bacterial core gene set, named UBCG2, that was selected from the more extensive genome sequence set belonging to 3,508 species spanning 43 phyla. UBCG2 comprises 81 genes with nine Clusters of Orthologous Groups of proteins (COGs) functional categories. The new gene set and complete pipeline are available at http://leb.snu.ac.kr/ubcg2.

Type 2 human papillomavirus E7 attenuates E-cadherin expression in human keratinocytes: Ji Young Song , Young Min Park , Soon Yong Choi; J. Microbiol. 2021;59(6):616-625. Published online March 29, 2021; DOI: https://doi.org/10.1007/s12275-021-0690-y

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Abstract: Human papillomaviruses (HPVs) are known to utilize the down-regulation of epithelial (E)-cadherin, a major component of adherens junctions of keratinocytes, to evade host immune surveillance in high-risk group. However, the effects of HPV on the function of E-cadherin in low-risk groups remain unknown. We investigated whether type 2 HPV (HPV- 2) E7 could induce alterations in E-cadherin expression in transiently transfected keratinocytes and cell lines expressing HPV-2 E7. To examine the expression pattern of E-cadherin in cutaneous warts and normal skin samples, immunohistochemical analysis was performed. Quantitative real-time polymerase chain reactions, luciferase assays, western blot, immunocytochemistry, and electron microscopy were used to evaluate the mRNA and protein expression levels of Ecadherin in normal human epidermal keratinocytes transfected with HPV-2 E7 plasmid DNA or E7-specific siRNA and in E7-expressing cell lines. E-cadherin expression levels in HPV-2 positive cutaneous warts were significantly decreased compared to those in normal skin (p < 0.05). Similarly, the mRNA and protein expression levels of E-cadherin in E7 transiently transfected cells were significantly decreased compared to those in empty vector-transfected cells. The decreases were restored by transfection with E7-specific siRNA (p < 0.05). Likewise, cell lines expressing E7 showed a decreased expression of E-cadherin. When the cells were cultured in low attachment plates, cell-to-cell aggregation was inhibited. Taken together, our data suggest that HPV-2 E7, the causative agent of cutaneous warts, could mediate the transcriptional repression of E-cadherin.

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