Journal of Microbiology

Volume 57(6); June 2019

Review

MINIREVIEW] Antisense peptide nucleic acids as a potential anti-infective agent: Hyung Tae Lee , Se Kye Kim , Jang Won Yoon; J. Microbiol. 2019;57(6):423-430. Published online May 27, 2019; DOI: https://doi.org/10.1007/s12275-019-8635-4

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27 Citations

Abstract: Antibiotics have long been used for anti-infective control of bacterial infections, growth promotion in husbandry, and prophylactic protection against plant pathogens. However, their inappropriate use results in the emergence and spread of multiple drug resistance (MDR) especially among various bacterial populations, which limits further administration of conventional antibiotics. Therefore, the demand for novel anti-infective approaches against MDR diseases becomes increasing in recent years. The peptide nucleic acid (PNA)- based technology has been proposed as one of novel antiinfective and/or therapeutic strategies. By definition, PNA is an artificially synthesized nucleic acid mimic structurally similar to DNA or RNA in nature and linked one another via an unnatural pseudo-peptide backbone, rendering to its stability in diverse host conditions. It can bind DNA or RNA strands complimentarily with high affinity and sequence specificity, which induces the target-specific gene silencing by inhibiting transcription and/or translation. Based on these unique properties, PNA has been widely applied for molecular diagnosis as well as considered as a potential anti-infective agent. In this review, we discuss the general features of PNAs and their application to various bacterial pathogens as new anti-infective or antimicrobial agents.

Journal Articles

A comprehensive in silico analysis of sortase superfamily: Adeel Malik , Seung Bum Kim; J. Microbiol. 2019;57(6):431-443. Published online May 27, 2019; DOI: https://doi.org/10.1007/s12275-019-8545-5

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Abstract: Sortases are cysteine transpeptidases that assemble surface proteins and pili in their cell envelope. Encoded by all Grampositive bacteria, few Gram-negative bacteria and archaea, sortases are currently divided into six classes (A-F). Due to the steep increase in bacterial genome data in recent years, the number of sortase homologues have also escalated rapidly. In this study, we used protein sequence similarity networks to explore the taxonomic diversity of sortases and also to evaluate the current classification of these enzymes. The resultant data suggest that sortase classes A, B, and D predominate in Firmicutes and classes E and F are enriched in Actinobacteria, whereas class C is distributed in both Firmicutes and Actinobacteria except Streptomyces family. Sortases were also observed in various Gram-negatives and euryarchaeota, which should be recognized as novel classes of sortases. Motif analysis around the catalytic cysteine was also performed and suggested that the residue at 2nd position from cysteine may help distinguish various sortase classes. Moreover, the sequence analysis indicated that the catalytic arginine is highly conserved in almost all classes except sortase F in which arginine is replaced by asparagine in Actinobacteria. Additionally, class A sortases showed higher structural variation as compared to other sortases, whereas inter-class comparisons suggested structures of class C and D2 exhibited best similarities. A better understanding of the residues highlighted in this study should be helpful in elucidating their roles in substrate binding and the sortase function, and successively could help in the development of strong sortase inhibitors.

Paracoccus jeotgali sp. nov., isolated from Korean salted and fermented shrimp: Juseok Kim , Joon Yong Kim , Hye Seon Song , In-Tae Cha , Seong Woon Roh , Se Hee Lee; J. Microbiol. 2019;57(6):444-449. Published online May 27, 2019; DOI: https://doi.org/10.1007/s12275-019-8704-8

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Abstract: A Gram-stain-negative and facultatively aerobic bacterium, designated as strain CBA4604T, was isolated from a traditional Korean salted and fermented shrimp food (saeu-jeot). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CBA4604T formed a clearly distinct phyletic lineage from closely related species within the genus Paracoccus. Strain CBA4604T was the most closely related to P. koreensis Ch05T (97.5% 16S rRNA gene sequence similarity) and other type strains (≤ 97.0%). The genome comprised a chromosome and two plasmids of 3,299,166 bp with 66.5% G+C content. The DNA-DNA relatedness values between strain CBA4604T and P. koreensis Ch05T, P. alcaliphilus DSM 8512T, and P. stylophorae KTW-16T were 30.5%, 22.9%, and 16.7%, respectively. Cells of the strain were short rod-shaped and oxidase- and catalase-positive. The growth of strain CBA- 4604T was observed at 10–40°C (optimum, 37°C), pH 6.0–10.0 (optimum, pH 7.0), and in the presence of 0–8.0% (w/v) NaCl (optimum, 0–2.0%). Strain CBA4604T contained ubiquinone 10 as the sole isoprenoid quinone and summed feature 8 (C18:1 ω7c/C18:1 ω6c) and C18:0 as the major cellular fatty acids. The polar lipids consisted of phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phospholipid, an unidentified aminolipid, an unidentified glycolipid, and three unidentified lipids. Based on its phylogenetic, genomic, phenotypic, and chemotaxonomic features, we concluded that strain CBA- 4604T represents a novel species in the genus Paracoccus and we propose the name Paracoccus jeotgali sp. nov. The type strain is CBA4604T (= KACC 19579T = JCM 32510T).

Cyanobacterial biodiversity of semiarid public drinking water supply reservoirs assessed via next-generation DNA sequencing technology: Adriana Sturion Lorenzi , Mathias Ahii Chia , Fabyano Alvares Cardoso Lopes , Genivaldo Gueiros Z. Silva , Robert A. Edwards , Maria do Carmo Bittencourt-Oliveira; J. Microbiol. 2019;57(6):450-460. Published online May 27, 2019; DOI: https://doi.org/10.1007/s12275-019-8349-7

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Abstract: Next-generation DNA sequencing technology was applied to generate molecular data from semiarid reservoirs during well-defined seasons. Target sequences of 16S-23S rRNA ITS and cpcBA-IGS were used to reveal the taxonomic groups of cyanobacteria present in the samples, and genes coding for cyanotoxins such as microcystins (mcyE), saxitoxins (sxtA), and cylindrospermopsins (cyrJ) were investigated. The presence of saxitoxins in the environmental samples was evaluated using ELISA kit. Taxonomic analyses of high-throughput DNA sequencing data showed the dominance of the genus Microcystis in Mundaú reservoir. Furthermore, it was the most abundant genus in the dry season in Ingazeira reservoir. In the rainy season, 16S-23S rRNA ITS analysis revealed that Cylindrospermopsis raciborskii comprised 46.8% of the cyanobacterial community in Ingazeira reservoir, while the cpcBAIGS region revealed that C. raciborskii (31.8%) was the most abundant taxon followed by Sphaerospermopsis aphanizomenoides (17.3%) and Planktothrix zahidii (16.6%). Despite the presence of other potential toxin-producing genera, the detected sxtA gene belonged to C. raciborskii, while the mcyE gene belonged to Microcystis in both reservoirs. The detected mcyE gene had good correlation with MC content, while the amplification of the sxtA gene was related to the presence of STX. The cyrJ gene was not detected in these samples. Using DNA analyses, our results showed that the cyanobacterial composition of Mundaú reservoir was similar in successive dry seasons, and it varied between seasons in Ingazeira reservoir. In addition, our data suggest that some biases of analysis influenced the cyanobacterial communities seen in the NGS output of Ingazeira reservoir.

Assembly mechanisms of soil bacterial communities in subalpine coniferous forests on the Loess Plateau, China: Pengyu Zhao , Jinxian Liu , Tong Jia , Zhengming Luo , Cui Li , Baofeng Chai; J. Microbiol. 2019;57(6):461-469. Published online May 27, 2019; DOI: https://doi.org/10.1007/s12275-019-8373-7

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Abstract: Microbial community assembly is affected by trade-offs between deterministic and stochastic processes. However, the mechanisms underlying the relative influences of the two processes remain elusive. This knowledge gap limits our ability to understand the effects of community assembly processes on microbial community structures and functions. To better understand community assembly mechanisms, the community dynamics of bacterial ecological groups were investigated based on niche breadths in 23 soil plots from subalpine coniferous forests on the Loess Plateau in Shanxi, China. Here, the overall community was divided into the ecological groups that corresponded to habitat generalists, ‘other taxa’ and specialists. Redundancy analysis based on Bray-Curtis distances (db-RDA) and multiple regression tree (MRT) analysis indicated that soil organic carbon (SOC) was a general descriptor that encompassed the environmental gradients by which the communities responded to, because it can explain more significant variations in community diversity patterns. The three ecological groups exhibited different niche optima and degrees of specialization (i.e., niche breadths) along the SOC gradient, suggesting the presence of a gradient in tolerance for environmental heterogeneity. The inferred community assembly processes varied along the SOC gradient, wherein a transition was observed from homogenizing dispersal to variable selection that reflects increasing deterministic processes. Moreover, the ecological groups were inferred to perform different community functions that varied with community composition, structure. In conclusion, these results contribute to our understanding of the trade-offs between community assembly mechanisms and the responses of community structure and function to environmental gradients.

Trophic strategy of diverse methanogens across a river-to-sea gradient: Bingchen Wang , Fanghua Liu , Shiling Zheng , Qinqin Hao; J. Microbiol. 2019;57(6):470-478. Published online May 27, 2019; DOI: https://doi.org/10.1007/s12275-019-8482-3

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Abstract: Methanogens are an important biogenic source of methane, especially in estuarine waters across a river-to-sea gradient. However, the diversity and trophic strategy of methanogens in this gradient are not clear. In this study, the diversity and trophic strategy of methanogens in sediments across the Yellow River (YR) to the Bohai Sea (BS) gradient were investigated by high-throughput sequencing based on the 16S rRNA gene. The results showed that the diversity of methanogens in sediments varied from multitrophic communities in YR samples to specific methylotrophic communities in BS samples. The methanogenic community in YR samples was dominated by Methanosarcina, while that of BS samples was dominated by methylotrophic Methanococcoides. The distinct methanogens suggested that the methanogenic community of BS sediments did not originate from YR sediment input. High-throughput sequencing of the mcrA gene revealed that active Methanococcoides dominated in the BS enrichment cultures with trimethylamine as the substrate, and methylotrophic Methanolobus dominated in the YR enrichment cultures, as detected to a limited amount in in situ sediment samples. Methanosarcina were also detected in this gradient sample. Furthermore, the same species of Methanosarcina mazei, which was widely distributed, was isolated from the area across a river-to-sea gradient by the culture-dependent
method
. In summary, our results showed that a distribution of diverse methanogens across a river-to-sea gradient may shed light on adaption strategies and survival mechanisms in methanogens.

Mixed starter of Lactococcus lactis and Leuconostoc citreum for extending kimchi shelf-life: Mi-Ju Kim , Hae-Won Lee , Mo-Eun Lee , Seong Woon Roh , Tae-Woon Kim; J. Microbiol. 2019;57(6):479-484. Published online May 27, 2019; DOI: https://doi.org/10.1007/s12275-019-9048-0

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Abstract: To develop a starter culture system for improving the shelflife and quality of kimchi, we prepared a mixed starter composed of Lactococcus lactis and Leuconostoc citreum. Two strains, L. lactis WiKim0098 and Leu. citreum WiKim0096, showed high antimicrobial activity and mannitol productivity, respectively. These lactic acid bacteria (LAB) were introduced as a starter into kimchi following cultivation in foodgrade liquid medium. Two kimchi samples, with and without starter, were fermented for 12 days at 10°C. Compared to the control kimchi without starter, a lower initial pH and higher number of LAB were observed in kimchi with starter at 0 day. However, the starter in kimchi prolonged the period taken by kimchi to reach to pH 4.2 by approximately 1.5-fold compared to that in the control kimchi. To estimate the effect of the starter on the flavor of kimchi, metabolite changes were evaluated by gas chromatography/mass spectrometry. In starter fermented kimchi, the levels of mannitol and amino acid, which are associated with the flavor of kimchi, were increased following fermentation. The amount of mannitol was confirmed by high-performance liquid chromatography analysis, showing concentrations of 3.4 and 5.1 mg/ml for the control and starter fermented kimchi, respectively. Thus, mixed starter inoculated with L. lactis WiKim0098 and Leu. citreum WiKim0096 may extend the shelf-life of kimchi and improve its sensory characteristics.

Biofilm characterization of Fusarium solani keratitis isolate: increased resistance to antifungals and UV light: Itzel Margarita Córdova-Alcántara , Diana Laura Venegas-Cortés , María Ángeles Martínez-Rivera , Néstor Octavio Pérez , Aida Verónica Rodriguez-Tovar; J. Microbiol. 2019;57(6):485-497. Published online May 27, 2019; DOI: https://doi.org/10.1007/s12275-019-8637-2

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42 Citations

Abstract: Fusarium solani has drawn phytopathogenic, biotechnological, and medical interest. In humans, it is associated with localized infections, such as onychomycosis and keratomycosis, as well as invasive infections in immunocompromised patients. One pathogenicity factor of filamentous fungi is biofilm formation. There is still only scarce information about the in vitro mechanism of the formation and composition of F. solani biofilm. In this work, we describe the biofilm formed by a clinical keratomycosis isolate in terms of its development, composition and susceptibility to different antifungals and ultraviolet light (UV) at different biofilm formation stages. We found five biofilm formation stages using scanning electron microscopy: adherence, germination, hyphal development, maturation, and cell detachment. Using epifluorescence microscopy with specific fluorochromes, it was elucidated that the extracellular matrix consists of carbohydrates, proteins, and extracellular DNA. Specific inhibitors for these molecules showed significant biofilm reductions. The antifungal susceptibility against natamycin, voriconazole, caspofungin, and amphotericin B was evaluated by metabolic activity and crystal violet assay, with the F. solani biofilm preformation to 24 h increased in resistance to natamycin, voriconazole, and caspofungin, while the biofilm preformation to 48 h increased in resistance to amphotericin B. The preformed biofilm at 24 h protected and reduced UV light mortality. F. solani isolate could produce a highly structured extra biofilm; its cellular matrix consists of carbohydrate polymers, proteins, and eDNA. Biofilm confers antifungal resistance and decreases its susceptibility to UV light. The fungal biofilm functions as a survival strategy against antifungals and environmental factors.

Novosphingobium sp. PP1Y as a novel source of outer membrane vesicles: Federica De Lise , Francesca Mensitieri , Giulia Rusciano , Fabrizio Dal Piaz , Giovanni Forte , Flaviana Di Lorenzo , Antonio Molinaro , Armando Zarrelli , Valeria Romanucci , Valeria Cafaro , Antonio Sasso , Amelia Filippelli , Alberto Di Donato , Viviana Izzo; J. Microbiol. 2019;57(6):498-508. Published online May 27, 2019; DOI: https://doi.org/10.1007/s12275-019-8483-2

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Abstract: Outer membrane vesicles (OMVs) are nanostructures of 20– 200 nm diameter deriving from the surface of several Gramnegative bacteria. OMVs are emerging as shuttles involved in several mechanisms of communication and environmental adaptation. In this work, OMVs were isolated and characterized from Novosphingobium sp. PP1Y, a Gram-negative non-pathogenic microorganism lacking LPS on the outer membrane surface and whose genome was sequenced and annotated. Scanning electron microscopy performed on samples obtained from a culture in minimal medium highlighted the presence of PP1Y cells embedded in an extracellular matrix rich in vesicular structures. OMVs were collected from the exhausted growth medium during the mid-exponential phase, and purified by ultracentrifugation on a sucrose gradient. Atomic force microscopy, dynamic light scattering and nanoparticle tracking analysis showed that purified PP1Y OMVs had a spherical morphology with a diameter of ca. 150 nm and were homogenous in size and shape. Moreover, proteomic and fatty acid analysis of purified OMVs revealed a specific biochemical “fingerprint”, suggesting interesting details concerning their biogenesis and physiological role. Moreover, these extracellular nanostructures do not appear to be cytotoxic on HaCaT cell line, thus paving the way to their future use as novel drug delivery systems.

Gastrointestinal microbiota alteration induced by Mucor circinelloides in a murine model: Katherine D. Mueller , Hao Zhang , Christian R. Serrano , R. Blake Billmyre , Eun Young Huh , Philipp Wiemann , Nancy P. Keller , Yufeng Wang , Joseph Heitman , Soo Chan Lee; J. Microbiol. 2019;57(6):509-520. Published online May 27, 2019; DOI: https://doi.org/10.1007/s12275-019-8682-x

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18 Citations

Abstract: Mucor circinelloides is a pathogenic fungus and etiologic agent of mucormycosis. In 2013, cases of gastrointestinal illness after yogurt consumption were reported to the US FDA, and the producer found that its products were contaminated with Mucor. A previous study found that the Mucor strain isolated from an open contaminated yogurt exhibited virulence in a murine systemic infection model and showed that this strain is capable of surviving passage through the gastrointestinal tract of mice. In this study, we isolated another Mucor strain from an unopened yogurt that is closely related but distinct from the first Mucor strain and subsequently examined if Mucor alters the gut microbiota in a murine host model. DNA extracted from a ten-day course of stool samples was used to analyze the microbiota in the gastrointestinal tracts of mice exposed via ingestion of Mucor spores. The bacterial 16S rRNA gene and fungal ITS1 sequences obtained were used to identify taxa of each kingdom. Linear regressions revealed that there are changes in bacterial and fungal abundance in the gastrointestinal tracts of mice which ingested Mucor. Furthermore, we found an increased abundance of the bacterial genus Bacteroides and a decreased abundance of the bacteria Akkermansia muciniphila in the gastrointestinal tracts of exposed mice. Measurements of abundances show shifts in relative levels of multiple bacterial and fungal taxa between mouse groups. These findings suggest that exposure of the gastrointestinal tract to Mucor can alter the microbiota and, more importantly, illustrate an interaction between the intestinal mycobiota and bacteriota. In addition, Mucor was able to induce increased permeability in epithelial cell monolayers in vitro, which might be indicative of unstable intestinal barriers. Understanding how the gut microbiota is shaped is important to understand the basis of potential methods of treatment for gastrointestinal illness. How the gut microbiota changes in response to exposure, even by pathogens not considered to be causative agents of food-borne illness, may be important to how commercial food producers prevent and respond to contamination of products aimed at the public. This study provides evidence that the fungal microbiota, though understudied, may play an important role in diseases of the human gut.

Nano-encapsulation of naringinase produced by Trichoderma longibrachiatum ATCC18648 on thermally stable biopolymers for citrus juice debittering: Manal M. Housseiny , Heba I. Aboelmagd; J. Microbiol. 2019;57(6):521-531. Published online May 27, 2019; DOI: https://doi.org/10.1007/s12275-019-8528-6

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18 Citations

Abstract: Characteristics of naringinase nano-encapsulated forms on different carrier materials (chitosan and alginate polymers) were investigated in this study. Screening of twelve fungal isolates for naringinase production indicated that Trichoderma longibrachiatum was the most promising. Grapefruit rind was used as a substrate containing naringin for naringinase production. TEM micrographs showed that chitosan nano-capsules were applied for the production of morphologically homogeneous enzymatic nano-particles with high enzyme encapsulation efficiency, small asymmetric sizes (from 15.09 to 27.07 nm with the mean of 21.8 nm) and rough surfaces compared to nano-encapsulated naringinase in alginate which showed nano-particle size (from 33.37 to 51.01 nm with the mean of 43.03 nm). It was revealed that the highest naringinase activity was found in case of chitosan nano-capsule naringinase compared to alginate nano-capsule one. Thermogram analysis (TGA) showed that the free enzyme loses about 92% of its weight at approximately 110°C, while the nanoencapsulated ones show more stability at higher temperatures. Conclusively, the nano-capsulation process improves the kinetics and operational stability so could be useful as a debittering agent for various thermal processing applications in citrus juices industries which makes the fruit juice more acceptable and cost-effective to the consumer.

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.

Published Erratum

Erratum] Bacillus piscis sp. nov., a novel bacterium isolated from the muscle of the antarctic fish Dissostichus mawsoni: Jae-Bong Lee , Seon Hwa Jeon , Seok-Gwan Choi , Hee-Young Jung , Myung Kyum Kim , Sathiyaraj Srinivasan; J. Microbiol. 2019;57(6):539-539.; DOI: https://doi.org/10.1007/s12275-019-0719-7

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Abstract: In the article by Lee et al. published in Journal of Microbiology 2016; 54, 809–813, The KCTC accession number KCTC 18866T in abstract and foot note should be corrected to KCTC 33839T. The sentence in abstract should have read: Based on the phylogenetic, phenotypic, and chemotaxonomic data, strain 16MFT21T (=KCTC 33839T =JCM 31664T). The species description in foot note should have read: The NCBI GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 16MFT21T (=KCTC 33839T =JCM 31664T) is KX753358. We apologize for any inconvenience that this may have caused.

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