Microbes and Environments
Online ISSN : 1347-4405
Print ISSN : 1342-6311
ISSN-L : 1342-6311
Volume 32 , Issue 3
Showing 1-15 articles out of 15 articles from the selected issue
Research Highlight
Minireview
  • Miho Yoshikawa, Ming Zhang, Koki Toyota
    Type: Minireview
    2017 Volume 32 Issue 3 Pages 188-200
    Published: 2017
    Released: September 27, 2017
    [Advance publication] Released: September 12, 2017
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    Volatile organic compounds (VOCs) are major pollutants that are found in contaminated sites, particularly in developed countries such as Japan. Various microorganisms that degrade individual VOCs have been reported, and genomic information related to their phylogenetic classification and VOC-degrading enzymes is available. However, the biodegradation of multiple VOCs remains a challenging issue. Practical sites, such as chemical factories, research facilities, and illegal dumping sites, are often contaminated with multiple VOCs. In order to investigate the potential of biodegrading multiple VOCs, we initially reviewed the biodegradation of individual VOCs. VOCs include chlorinated ethenes (tetrachloroethene, trichloroethene, dichloroethene, and vinyl chloride), BTEX (benzene, toluene, ethylbenzene, and xylene), and chlorinated methanes (carbon tetrachloride, chloroform, and dichloromethane). We also summarized essential information on the biodegradation of each kind of VOC under aerobic and anaerobic conditions, together with the microorganisms that are involved in VOC-degrading pathways. Interactions among multiple VOCs were then discussed based on concrete examples. Under conditions in which multiple VOCs co-exist, the biodegradation of a VOC may be constrained, enhanced, and/or unaffected by other compounds. Co-metabolism may enhance the degradation of other VOCs. In contrast, constraints are imposed by the toxicity of co-existing VOCs and their by-products, catabolite repression, or competition between VOC-degrading enzymes. This review provides fundamental, but systematic information for designing strategies for the bioremediation of multiple VOCs, as well as information on the role of key microorganisms that degrade VOCs.

Regular Papers
  • Ruizhen Wang, Leiming Dong, Yuequ Chen, Liangjian Qu, Qinghua Wang, Yo ...
    Type: Regular Papers
    2017 Volume 32 Issue 3 Pages 201-209
    Published: 2017
    Released: September 27, 2017
    [Advance publication] Released: August 19, 2017
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    Symbioses have played pivotal roles in biological, ecological, and evolutionary diversification. Symbiotic bacteria affect the biology of hosts in a number of ways. Esteya vermicola, an endoparasitic nematophagous fungus, has high infectivity in the pine wood nematode (PWN), which causes devastating ecological damage and economic losses in Asia and Europe. An integration of molecular, phylogenetic, and morphological analyses revealed that surface-sterilized E. vermicola with septate hyphae from different geographic locations harbor bacterial endosymbionts. 16S rRNA gene sequences from four fungal strains all clustered in a well-supported monophyletic clade that was the most closely related to Pseudomonas stutzeri and affiliated with Gammaproteobacteria. The existence and intracellular location of endobacteria was revealed by fluorescent in situ hybridization (FISH). Our results showed that endobacteria were coccoid, vertically inherited, as yet uncultured, and essential symbionts. Ultrastructural observations indicated that young and old endobacteria differed in cell size, cell wall thickness, and the degree of reproduction. The results of the present study provide a fundamental understanding of the endobacteria inside E. vermicola and raise questions regarding the impact of endobacteria on the biology, ecology, and evolution of their fungal host.

  • Léa Girard, Sébastien Peuchet, Pierre Servais, Annabelle Henry, Nadine ...
    Type: Regular Papers
    2017 Volume 32 Issue 3 Pages 210-218
    Published: 2017
    Released: September 27, 2017
    [Advance publication] Released: July 19, 2017
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    A cellular approach combining Direct Viable Counting and Fluorescent In Situ Hybridization using a one-step multiple-probe technique and Solid Phase Cytometry (DVC-FISH-SPC) was developed to monitor total viable vibrios and cover the detection of a large diversity of vibrios. FISH combined three probes in the same assay and targeted sequences located at different positions on the 16S rRNA of Vibrio and Aliivibrio members. We performed a 10-month in situ study to investigate the weekly dynamics of viable vibrios relative to culturable counts at two northwestern Mediterranean coastal sites, and identified the key physicochemical factors for their occurrence in water using a multivariate analysis. Total viable and culturable cell counts showed the same temporal pattern during the warmer season, whereas the ratios between both methods were inverted during the colder seasons (<15°C), indicating that some of the vibrio community had entered into a viable but non-culturable (VBNC) state. We confirmed that Seawater Surface Temperature explained 51–62% of the total variance in culturable counts, and also showed that the occurrence of viable vibrios is controlled by two variables, pheopigment (15%) and phosphate (12%) concentrations, suggesting that other unidentified factors play a role in maintaining viability.

  • Iulia Lupan, Rahela Carpa, Andreea Oltean, Beatrice Simona Kelemen, Oc ...
    Type: Regular Papers
    2017 Volume 32 Issue 3 Pages 219-225
    Published: 2017
    Released: September 27, 2017
    [Advance publication] Released: August 05, 2017
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    The occurrence and spread of bacterial antibiotic resistance are subjects of great interest, and the role of wastewater treatment plants has been attracting particular interest. These stations are a reservoir of bacteria, have a large range of organic and inorganic substances, and the amount of bacteria released into the environment is very high. The main purpose of the present study was to assess the removal degree of bacteria with resistance to antibiotics and identify the contribution of a wastewater treatment plant to the microbiota of Someşul Mic river water in Cluj county. The resistance to sulfamethoxazole and tetracycline and some of their representative resistance genes: sul1, tet(O), and tet(W) were assessed in this study. The results obtained showed that bacteria resistant to sulphonamides were more abundant than those resistant to tetracycline. The concentration of bacteria with antibiotic resistance changed after the treatment, namely, bacteria resistant to sulfamethoxazole. The removal of all bacteria and antibiotic-resistant bacteria was 98–99% and the degree of removal of bacteria resistant to tetracycline was higher than the bacteria resistant to sulfamethoxazole compared to total bacteria. The wastewater treatment plant not only contributed to elevating ARG concentrations, it also enhanced the possibility of horizontal gene transfer (HGT) by increasing the abundance of the intI1 gene. Even though the treatment process reduced the concentration of bacteria by two orders of magnitude, the wastewater treatment plant in Cluj-Napoca contributed to an increase in antibiotic-resistant bacteria concentrations up to 10 km downstream of its discharge in Someşul Mic river.

  • Gitanjali NandaKafle, Tarren Seale, Toby Flint, Madhav Nepal, Stephanu ...
    Type: Regular Papers
    2017 Volume 32 Issue 3 Pages 226-233
    Published: 2017
    Released: September 27, 2017
    [Advance publication] Released: July 27, 2017
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    Escherichia coli is widely considered to not survive for extended periods outside the intestines of warm-blooded animals; however, recent studies demonstrated that E. coli strains maintain populations in soil and water without any known fecal contamination. The objective of this study was to investigate whether the niche partitioning of E. coli occurs between cattle and their pasture. We attempted to clarify whether E. coli from bovine feces differs phenotypically and genotypically from isolates maintaining a population in pasture soil over winter. Soil, bovine fecal, and run-off samples were collected before and after the introduction of cattle to the pasture. Isolates (363) were genotyped by uidA and mutS sequences and phylogrouping, and evaluated for curli formation (Rough, Dry, And Red, or RDAR). Three types of clusters emerged, viz. bovine-associated, clusters devoid of cattle isolates and representing isolates endemic to the pasture environment, and clusters with both. All isolates clustered with strains of E. coli sensu stricto, distinct from the cryptic species Clades I, III, IV, and V. Pasture soil endemic and bovine fecal populations had very different phylogroup distributions, indicating niche partitioning. The soil endemic population was largely comprised of phylogroup B1 and had a higher average RDAR score than other isolates. These results indicate the existence of environmental E. coli strains that are phylogenetically distinct from bovine fecal isolates, and that have the ability to maintain populations in the soil environment.

  • Qian-Shan Qin, Ding-Shan Feng, Peng-Fei Liu, Qiao He, Xia Li, Ai-Ming ...
    Type: Regular Papers
    2017 Volume 32 Issue 3 Pages 234-243
    Published: 2017
    Released: September 27, 2017
    [Advance publication] Released: August 05, 2017
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    The methanogenic degradation of hydrocarbons plays an important role in hydrocarbon-contaminated environments in the absence of an external electron acceptor. Members of Syntrophaceae sublineages were previously reported to be responsible for syntrophic alkane degradation. However, limited information is currently available on their physiological capabilities in nature because it is very challenging to cultivate these as-yet uncultured microbes. We herein performed metagenomic sequencing of the methanogenic hexadecane-degrading culture M82 and recovered a nearly complete genome (2.75 Mb, estimated completeness ≥97%) belonging to Syntrophaceae sublineage II. The assembly genome was tentatively named “Candidatus Smithella cisternae strain M82_1”. Genes encoding alkylsuccinate synthase for alkane activation were identified, suggesting that this organism is capable of oxidizing alkanes through fumarate addition. This capability was further supported by the detection of methyl pentadecyl succinic acid and methyl tetradecyl succinic acid in cultures amended with hexadecane and pentadecane, respectively. Genes encoding enzymes for the β-oxidation of long-chain fatty acids and butyrate were also identified. The electron transfer flavoprotein/DUF224 complex is presumed to link electron flow from acyl-CoA dehydrogenase to a membrane hydrogenase or formate dehydrogenase. Although no indications of Rnf complexes were detected, genes encoding electron-confurcating hydrogenase and formate dehydrogenase were proposed to couple the thermodynamically favorable oxidation of ferredoxin to generate H2 and formate from NADH. Strain M82_1 synthesized ATP from acetyl-CoA by substrate-level phosphorylation or F1F0-ATP synthases. These results provide an insight into the potential metabolic traits and ecophysiological roles of the syntrophic alkane degrader Syntrophaceae.

  • María del Rocío Bustillos-Cristales, Ivan Corona-Gutierrez, Miguel Cas ...
    Type: Regular Papers
    2017 Volume 32 Issue 3 Pages 244-251
    Published: 2017
    Released: September 27, 2017
    [Advance publication] Released: August 30, 2017
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    Methanol-consuming culturable bacteria were isolated from the plant surface, rhizosphere, and inside the stem of Neobuxbaumia macrocephala. All 38 isolates were facultative methylotrophic microorganisms. Their classification included the Classes Actinobacteria, Sphingobacteriia, Alpha-, Beta-, and Gammaproteobacteria. The deduced amino acid sequences of methanol dehydrogenase obtained by PCR belonging to Actinobacteria, Alpha-, Beta-, and Gammaproteobacteria showed high similarity to rare-earth element (REE)-dependent XoxF methanol dehydrogenases, particularly the group XoxF5. The sequences included Asp301, the REE-coordinating amino acid, present in all known XoxF dehydrogenases and absent in MxaF methanol dehydrogenases. The quantity of the isolates showed positive hybridization with a xoxF probe, but not with a mxaF probe. Isolates of all taxonomic groups showed methylotrophic growth in the presence of Ce3+ or Ca2+. The presence of xoxF-like sequences in methylotrophic bacteria from N. macrocephala and its potential relationship with their adaptability to xerophytic plants are discussed.

  • Flore Chirila, Alexandra Tabaran, Nicodim Fit, George Nadas, Marian Mi ...
    Type: Regular Papers
    2017 Volume 32 Issue 3 Pages 252-259
    Published: 2017
    Released: September 27, 2017
    [Advance publication] Released: September 12, 2017
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    This study was conducted in order to assess the antimicrobial resistance patterns of E. coli isolated from young animals affected between 1980 and 2016. The selected isolates for this study (n=175) carried stx1/stx2 genes and the most prevalent type of pathogenic E. coli found belonged to serogroup O101, antigen (K99)–F41 positive. All STEC-positive isolates were tested for susceptibility to 11 antimicrobials. Multidrug resistance (MDR) increased from 11% during the 1980s to 40% between 2000 and 2016. Resistance to tetracycline and streptomycin was the most frequent co-resistance phenotype (37%). Co-resistance to tetracycline and sulfonamide was found in 21% of E. coli isolates, while the MDR pattern to tetracycline, sulfonamide, and streptomycin was observed in 12% of the strains tested. Only 8% of isolates were co-resistant to tetracycline, ampicillin, streptomycin, and sulfonamide. The most common resistance genes found were those encoding for tetracycline, sulphonamides, and streptomycin, with 54% (n=95) of the tested isolates containing at least one of the genes encoding tetracycline resistance. A total of 87% of E. coli that tested positive for tetracycline (tetA, tetB, and tetC) and sulphonamide (sul1) resistance genes were isolated between 2000 and 2016. A large number of isolates (n=21) carried int1 and a nucleotide sequence analysis revealed that all class 1 integron gene cassettes carried sul1, tet, and dfrA1 resistance genes. An increase was observed in the level of resistance to antimicrobials in Romania, highlighting the urgent need for a surveillance and prevention system for antimicrobial resistance in livestock in Eastern Europe.

  • Awaluddin Nurmiyanto, Hiroya Kodera, Tomonori Kindaichi, Noriatsu Ozak ...
    Type: Regular Papers
    2017 Volume 32 Issue 3 Pages 260-267
    Published: 2017
    Released: September 27, 2017
    [Advance publication] Released: September 09, 2017
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    Candidatus Accumulibacter phosphatis (Accumulibacter), which plays an important role in enhanced biological phosphorus removal in wastewater treatment plants, is phylogenetically classified into two major types (Types I and II). Phosphate concentrations affect the Accumulibacter community of the biomass enriched in treatment plants. Therefore, in the present study, Accumulibacter enrichments were conducted using a down-flow hanging sponge reactor under five conditions and a wide range of controlled phosphate concentrations in order to investigate how phosphate governs the community. We found that excessive phosphate levels inhibited Accumulibacter activity, that this inhibitory effect was greater for Type II. In addition, the affinity of Type II for phosphate was higher than that of Type I. Type IIA-B dominated at a phosphate concentration less than 5 mg P L−1, while Type IA was dominant at 50 and 500 mg P L−1. These patterns of enrichment may be explained by an inhibition kinetics model.

  • Sachi Tanaka, Kana Yamamoto, Chisato Hamajima, Fuka Takahashi, Kazuki ...
    Type: Regular Papers
    2017 Volume 32 Issue 3 Pages 268-274
    Published: 2017
    Released: September 27, 2017
    [Advance publication] Released: September 12, 2017
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    We aimed to investigate the effects of feeding fermented Brassica rapa L. on ecological and immunological changes in the mouse gut using in vitro cultivation tests and in vivo experiments in normal mice. In the preliminary in vitro study, two B. rapa L. products from different fermentation periods (one d [SF] or six months [LF]) were evaluated along with non-fermented vegetables (NF). Among the components of each product, the insoluble fraction resulted in the most prominent change such as a relative increase in butyrate production during a cultivation inoculated with mouse cecum contents. Based on this result, the boiled water-insoluble fractions of B. rapa L. (SF, LF, and NF samples) were selected as test materials for the subsequent in vivo experiment. Male C57BL/6J mice were divided into four groups and fed either a control diet (CON) or control diet plus one of the insoluble fractions for two weeks. The NF and LF groups had higher relative populations of Faecalibacterium prausnitzii than the CON group. Therefore, colonic butyrate concentrations were higher in the NF and LF groups than in the CON group. The oral administration of B. rapa L. extract induced immune regulatory effects, even when mice were fed NF and SF, but not LF, as assessed by an increase in regulatory T cell numbers. Our results indicate that feeding a purified insoluble fraction from B. rapa L. affects enteric short-chain fatty acid production and immunological responses in the mouse gut in a similar manner, regardless of the fermentation status.

  • Ana Umpiérrez, Inés Bado, Martín Oliver, Sofía Acquistapace, Analía Et ...
    Type: Regular Papers
    2017 Volume 32 Issue 3 Pages 275-282
    Published: 2017
    Released: September 27, 2017
    [Advance publication] Released: September 12, 2017
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    Escherichia coli is one of the main etiological agents of neonatal calf diarrhea (NCD). The objective of this study was to assess the presence of virulence genes, genetic diversity, and antibiotic resistance mechanisms in E. coli associated with NCD in Uruguay. PCR was used to assess the presence of intimin, Shiga-like toxin, and stable and labile enterotoxin genes. Resistance to fluoroquinolones and oxyimino-cephalosporins was estimated on Müller-Hinton agar plates. Further antibiotic disc-diffusion tests were performed to assess bacterial multi-resistance. The presence of PMQR, ESBL, MCR-1, and integron genes was evaluated. Isolates were typed using ERIC-PCR, and 20 were selected for MLST, adhesion to Hep-2 cells, in vitro biofilm formation, and eukaryotic cytotoxicity. The prevalence of ETEC genes was lower than 3% in each case (estA and elt). Six isolates were EPEC (eae+) and 2 were EHEC/STEC (eae+/stx1+). The results of a diversity analysis showed high genetic heterogenicity among isolates. Additionally, different sequence types, including ST10, ST21, and ST69, were assigned to selected isolates. Thirty-six percent (96/264) of the isolates were fluoroquinolone-resistant, with 61/96 (63.5%) being multidrug-resistant. Additionally, 6 were oxyimino-cephalosporin-resistant. The qnrB, qnrS1, and blaCTX-M-14 genes were detected, whereas no isolates carried the mcr-1 gene. Isolates had the ability to adhere to Hep-2 cells and form biofilms. Only 1 isolate expressed toxins in vitro. E. coli from NCD cases in Uruguay are very diverse, potentially virulent, and may interact with eukaryotic cells. Zoonotic potential, together with resistance traits and the presence of horizontal transfer mechanisms, may play a significant role in infections caused by these microorganisms.

Short Communications
  • Hiroki Suga, Sakiko Kikuchi, Yasuo Takeichi, Chihiro Miyamoto, Masaaki ...
    Type: Short Communication
    2017 Volume 32 Issue 3 Pages 283-287
    Published: 2017
    Released: September 27, 2017
    [Advance publication] Released: August 05, 2017
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    Natural bacteriogenic iron oxides (BIOS) were investigated using local-analyzable synchrotron-based scanning transmission X-ray microscopy (STXM) with a submicron-scale resolution. Cell, cell sheath interface (EPS), and sheath in the BIOS were clearly depicted using C-, N-, and O- near edge X-ray absorption fine structure (NEXAFS) obtained through STXM measurements. Fe-NEXAFS obtained from different regions of BIOS indicated that the most dominant iron mineral species was ferrihydrite. Fe(II)- and/or Fe(III)-acidic polysaccharides accompanied ferrihydrite near the cell and EPS regions. Our STXM/NEXAFS analysis showed that Fe species change continuously between the cell, EPS, and sheath under several 10-nm scales.

  • Yasuhiro Tanaka, Hiroaki Matsuzawa, Hideyuki Tamaki, Masahiro Tagawa, ...
    Type: Short Communication
    2017 Volume 32 Issue 3 Pages 288-292
    Published: 2017
    Released: September 27, 2017
    [Advance publication] Released: July 22, 2017
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    A number of novel bacteria including members of rarely cultivated phyla, Acidobacteria and Verrucomicrobia, were successfully isolated from the roots of two emergent plants, Iris pseudacorus and Scirpus juncoides, by a simple culturing method. A total of 47.1% (66 strains) for I. pseudacorus and 42.1% (59 strains) for S. juncoides of all isolates (140 strains from each sample) were phylogenetically novel. Furthermore, Acidobacteria and Verrucomicrobia occupied 10.7% (15 strains) and 2.9% (4 strains) of I. pseudacorus isolates, and 2.1% (3 strains) and 3.6% (5 strains) of S. juncoides isolates, respectively, indicating that plant roots are attractive sources for isolating rarely cultivated microbes.

  • Kanit Assawatheptawee, Uttapoln Tansawai, Anong Kiddee, Phetrada Thong ...
    Type: Short Communication
    2017 Volume 32 Issue 3 Pages 293-296
    Published: 2017
    Released: September 27, 2017
    [Advance publication] Released: September 09, 2017
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    Sixty-eight cefotaxime-resistant Escherichia coli isolates were recovered from different water environments in Northern Thailand. Isolates were mostly resistant to ceftazidime and aztreonam (>90%). The most common extended-spectrum β-lactamase-encoding gene was blaCTX-M-group 1 (75%) followed by blaCTX-M-group 9 (13.2%). The co-existence of blaCTX-M and AmpC-type β-lactamase genes was detected in 4 isolates (5.9%). Two E. coli isolates carrying blaCTX-M from canal and river water samples belonged to the phylogenetic group B2-ST131, which is known to be pathogenic. This is the first study on blaCTX-M and blaCMY-2-carrying E. coli and the emergence of ST131 from water environments in Thailand.

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