Enterobacter Ludwigii Research Articles

A dual approach employing MALDI-TOF MS and real-time PCR for fast species identification within the Enterobacter cloacae complex

A real-time PCR procedure targeting the gene of the molecular cochaperon DnaJ (dnaJ) was developed for specific detection of strains belonging to the Enterobacter cloacae group. The inclusivity and exclusivity of the real-time PCR assay were assessed with seven reference strains of E.cloacae, 12 other Enterobacter species and 41 non-Enterobacter strains. Inclusivity as well as exclusivity of the duplex real-time PCR was 100%. In contrast, resolution of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was inadequate for delineation of Enterobacter asburiae, Enterobacter hormaechei, Enterobacter kobei and Enterobacter ludwigii from E.cloacae. Eleven of 56 (20%) clinical isolates of the E.cloacae group could not be clearly identified as a certain species using MALDI-TOF MS. In summary, the combination of MALDI-TOF MS with the E.cloacae-specific duplex real-time PCR is an appropriate method for identification of the six species of the E.cloacae complex.

凉水保护区土壤产类漆酶-多铜氧化酶细菌群落结构

Laccases catalyze the oxidation of various aromatics,particularly phenolic and amine substrates,making them valuable in industrial applications.Laccases also play an important role in soil organic matter(SOM) turnover processes and the global carbon cycle due to their involvement in the synthesis and degradation of lignin as well as transformation of lignified substrates and humic substances.Laccases belong to the protein family of multicopper oxidases characterized by copper atoms in the active center.Laccases or laccase-like multicopper oxidases(LMCO) have been extensively studied especially in fungi.Recently,increasing evidence points to a wide occurrence of LMCO in bacteria.As bacterial communities are known to decompose pollutants and municipal wastes involving large quantities of phenolic substances and organic matter,it can be deduced that bacterial LMCO might also participate in lignin degradation and SOM cycling.Copper atoms not only constitute the catalytic center of laccases but can also confer copper resistance to bacteria.Consequently,copper ions have a well known use in isolating LMCO-producing bacteria.To gain deeper insights into the ecological role of bacteria with LMCO activity in soil,we used traditional plating methods and PCR-DGGE(denaturing gradient gel electrophoresis) technology to examine the community structure of LMCO-producing bacteria in forest soil of Liangshui nature reserve,Heilongjiang Province. Soil samples were collected in three patterns of forests: Pinus koraiensis,Betula platyphylla and Picea dietrich.Soil samples were enriched in M9 basal medium and then screened in LB medium with gradient Cu2+.Ten bacteria strains were isolated and identified based on traditional plating methods of bacteria separation.The results showed that they all belonged to Bacillus sp.,falling into four different species: Bacillus pumilus,Bacillus subtilis,Bacillus fusiformis and Bacillus amyloliquefaciens,among which no laccase-relative results have been reported on Bacillus fusiformis.To investigate the structural variation of LMCO-producing bacteria,16S-rDNA-V3-fragment-based DGGE and clone sequence BLAST technologies were applied.The data based on DGGE plot indicated there was no distinct variation of LMCO-producing bacteria structure among soil samples of different forest patterns in the research district.The diversity of LMCO-producing bacteria in Pinus koraiensis forests was more abundant than that in Betula platyphylla and Picea dietrich.The results derived from BLAST revealed that Ralstonia sp.,Enterobacter sp.and other uncultured species existed in soil samples in addition to Bacillus sp..Band 3 was present in all soil samples and had a 100% homology to Ralstonia mannitolilytica.Ralstonia sp.had highly physical and biochemical similarities as well as sequence homology with Cupriavidus sp.which was a copper-philic bacteria community.Bands 2,7 and 11 were commonly present except lane 8 and had a highly homology with Enterobacter ludwigii which had recently attracted interest in environment ecological research.Bands 5 and 9 had a highly homology with Bacillus circulans and Bacillus thuringiensis,respectively.Furthermore,Bands 1,4,6,8 and 10 had highly homology with an uncultured bacterial clone which needed further investigation.

A Multiphasic Approach for the Identification of Endophytic Bacterial in Strawberry Fruit and their Potential for Plant Growth Promotion

This study used a multiphasic approach, characterized by the simultaneous use of culture-dependent and culture-independent methods, to investigate endophytic bacterial communities in strawberry (Fragaria ananassa) fruit. A total of 92 bacterial endophytes were isolated and initially grouped by their repetitive extragenic palindromic (rep)-PCR banding pattern and biochemical features. Phylogenetic analysis of the 16S rRNA gene sequences of 45 representatives showed that the isolates belonged to the species Bacillus subtilis (eight isolates), Bacillus sp. (seven isolates), Enterobacter sp. (seven isolates), Enterobacter ludwigii (six isolates), Lactobacillus plantarum (six isolates), Pseudomonas sp. (five isolates), Pantoea punctata (three isolates), and Curtobacterium citreum (three isolates). Nucleic acids were extracted from the strawberry fruit and subjected to 16S rRNA gene directed polymerase chain reaction denaturing gradient gel electrophoresis (16S rRNA PCR-DGGE). The species B. subtilis, Enterobacter sp., and Pseudomonas sp. were detected both by isolation and DGGE. The DGGE fingerprints of total bacterial DNA did not exhibit bands corresponding to several of the representative species isolated in the extinction dilution (L. plantarum, C. citreum, and P. punctata). In contrast, bands in the DGGE profile that were identified as relatives of Arthrobacter sp. and one uncultivable Erythrobacter sp. were not recovered by cultivation techniques. After isolation, the nitrogen fixation ability and the in vitro production of indole-3-acetic acid (IAA) equivalents and siderophores were evaluated. A high percentage of isolates were found to possess the ability to produce siderophores and IAA equivalents; however, only a few isolates belonging to the genera Pseudomonas and Enterobacter showed the ability to fix nitrogen. Plant growth promotion was evaluated under greenhouse conditions and revealed the ability of the Bacillus strains to enhance the number of leaves, shoot length, root dry weight, and shoot dry weight. The activity of the bacterial isolate identified as B. subtilis NA-108 exerted the greatest influence on strawberry growth and showed a 42.8% increase in number of leaves, 15.26% for high shoot, 43.5% increase in root dry weight, and a 77% increase in shoot dry weight when compared with untreated controls.

Isolation of tannase-producing Enterobacter ludwigii GRT-1 from the rumen of migratory goats

We report for the first time isolation and characterization of a tannase-producing anaerobe from the rumen of migratory goats browsing on forage containing the anti-nutritional hydrolysable tannin. A total of sixteen bacterial isolates were isolated. The isolates were capable of degrading tannic acid to gallic acid and pyrogallol. One of the isolates due to its unique tolerance up to a level of 3.5% (w/v) of tannic acid, and production of tannase was further investigated and identified as Enterobacter ludwigii GRT-1, based on 16S rRNA gene sequencing.

Hydrocarbon degradation, plant colonization and gene expression of alkane degradation genes by endophytic Enterobacter ludwigii strains

The genus Enterobacter comprises a range of beneficial plant-associated bacteria showing plant growth promotion. Enterobacter ludwigii belongs to the Enterobacter cloacae complex and has been reported to include human pathogens but also plant-associated strains with plant beneficial capacities. To assess the role of Enterobacter endophytes in hydrocarbon degradation, plant colonization, abundance and expression of CYP153 genes in different plant compartments, three plant species (Italian ryegrass, birdsfoot trefoil and alfalfa) were grown in sterile soil spiked with 1% diesel and inoculated with three endophytic E. ludwigii strains. Results showed that all strains were capable of hydrocarbon degradation and efficiently colonized the rhizosphere and plant interior. Two strains, ISI10-3 and BRI10-9, showed highest degradation rates of diesel fuel up to 68% and performed best in combination with Italian ryegrass and alfalfa. All strains expressed the CYP153 gene in all plant compartments, indicating an active role in degradation of diesel in association with plants.

Bacterial species associated with sound and Botrytis-infected grapes from a Greek vineyard

Grape bacterial microbiota plays central roles in the quality of grapes and wine, yet its diversity remains poorly described. In the present study, bacterial species associated with sound and Botrytis-infected grapes of two cultivars originating from the same vineyard were assessed. Isolates were identified by PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and sequence analysis of partial 16S rRNA gene. Comparable counts were recorded between Botrytis-infected and sound grape samples. In all cases, the majority of isolates belonged to different species of Enterobacteriaceae. The dominant species in the vineyard was Klebsiella oxytoca that was found in different combinations with Citrobacter freundii, Enterobacter spp., Erwinia sp., Pantoea dispersa, Tatumella ptyseos or other species. In fermenting musts, those populations declined while other species evolved, like Lactobacillus plantarum and Enterobacter ludwigii. Populations in botrytised samples persisted longer during spontaneous fermentations. Present study suggests that bacterial diversity on grapes may be wider than previously described.

Comparison of microbial diversity of edible flowers and basil grown with organic versus conventional methods

The consumption and use of edible flowers as food is growing; however, no study has been conducted to evaluate their role in the cause of food-borne illness or in food safety. Recent food-borne outbreaks traced to fresh herbs have raised concern about their processing and handling. Basil, one of the most commonly used fresh herbs, has been identified as a source of food-borne illness. Baseline assessments of microflora were performed, and the microbial diversity between growing methods (organic vs. conventional) was compared. DNA sequencing was used to identify the microbial flora present on fresh edible flower and basil samples. The most predominant species identified were Enterobacter hormaechei (10%), Acinetobacter calcoaceticus (10%), Enterobacter ludwigii (10%), Enterobacter asburiae (6%), and Enterobacter cowanii (6%). Pseudomonas aeruginosa (6%), Salmonella enterica (6%), and Bacillus amyloliquefaciens (2%) were also isolated. Phylogenetic analysis showed that most species of isolated bacteria belonged to the phyla Gammaproteobacteria (81.2%) and Firmicutes (18.8%). Statistical analysis, diversity index for species richness, and lineage-per-time plots showed that for basil, organically grown samples had a higher microbial diversity than conventionally grown samples. Edible flowers and basil are often grown using organic methods and are commonly consumed raw without any washing or cooking, to preserve aesthetic value, but these practices may pose a potential risk for food-borne illness. The baseline assessment, together with phylogenetic and statistical analyses, indicated possible microbial contamination in edible flowers and basil. The use of statistical estimation of molecular diversity based on the 16S rRNA sequences and lineage-per-time plots with phylogenetic analysis well served as a means for comparing microbial diversity in food samples between the growing methods (organic vs. conventional).

Evaluation of bacteria isolated from rice rhizosphere for biological control of charcoal rot of sorghum caused by Macrophomina phaseolina (Tassi) Goid.

A total of 360 bacteria, isolated from the rhizospheres of a system of rice intensification (SRI) fields, were characterized for the production of siderophore, fluorescence, indole acetic acid (IAA), hydrocyanic acid (HCN) and solubilization of phosphorus. Of them, seven most promising isolates (SRI-156, -158, -178, -211, -229, -305 and -360) were screened for their antagonistic potential against Macrophomina phaseolina (causes charcoal rot in sorghum) by dual culture assay, blotter paper assay and in greenhouse. All the seven isolates inhibited M. phaseolina in dual culture assay, whereas six isolates solubilized phosphorous (except SRI-360), all seven produced siderophore, four produced fluorescence (except SRI-178, -229 and -305), six produced IAA (except SRI-305) and five produced HCN (except SRI-158 and -305). In the blotter paper assay, no charcoal rot infection was observed in SRI-156-treated sorghum roots, indicating complete inhibition of the pathogen, while the roots treated with the other isolates showed 49-76% lesser charcoal rot infection compared to the control. In the antifungal activity test (in green house on sorghum), all the isolates increased shoot dry mass by 15-23% and root dry mass by 15-20% (except SRI-158 and -360), over the control. In order to confirm the plant growth-promoting (PGP) traits of the isolates, the green house experiment was repeated but, in the absence of M. phaseolina. The results further confirmed the PGP traits of the isolates as evidenced by increases in shoot and root dry mass, 22-100% and 5-20%, respectively, over the control. The sequences of 16S rDNA gene of the isolates SRI-156, -158, -178, -211, -229, -305 and -360 were matched with Pseudomonas plecoglossicida, Brevibacterium antiquum, Bacillus altitudinis, Enterobacter ludwigii, E. ludwigii, Acinetobacter tandoii and P. monteilii, respectively in BLAST analysis. This study indicates that the selected bacterial isolates have the potential for PGP and control of charcoal rot disease in sorghum.

Exopolysaccharide production by nitrogen-fixing bacteria within nodules of Medicago plants exposed to chronic radiation in the Chernobyl exclusion zone

Nitrogen-fixing bacteria isolated from root nodules of Medicago plants growing in the 10 km zone around the Chernobyl nuclear power plant were screened for the production of new water-soluble acidic exopolysaccharides (EPSs). The different strains belonged to the Enteriobacteriaceae family ( Enterobacter ludwigii, Raoultella terrigena, Klebsiella oxytoca), except for one which belonged to the Rhizobiaceae family ( Sinorhizobium meliloti). All of the bacteria produced highly viscous EPS with an average molecular weight comprised between 1 × 10 6 and 3 × 10 6 Da. Five different compositions of EPS were characterized by physico-chemical analyses and 1H NMR spectroscopy: galactose/mannose (2/1), galactose/glucose (1/1), galactose/glucose/mannose (1/2/1), fucose/galactose/glucose (2/1/1) and fucose/galactose/glucose/mannose (2/2/1/1 or 1/1/2/4). Glucuronic acid, a charged monosaccharide, was also recovered in most of the different EPSs.

Establishment of long-term proliferating shoot cultures of elite Jatropha curcas L. by controlling endophytic bacterial contamination

Leaf explants of the second or third node were collected from field-grown elite Jatropha curcas trees and incubated in Murashige and Skoog’s (Physiol Plant 15:473–497, 1962) medium supplemented with growth regulators. Direct shoot organogenesis was induced when explants were incubated in a medium containing 0.5 mg l−1 benzyladenine (BA) and 0.1 mg l−1 indolebutyric acid (IBA). A maximum of seven shoot buds differentiated within 6 weeks of culture incubation. Indirect shoot organogenesis was obtained when explants were incubated in the medium supplemented with 0.5 mg l−1 BA along with 1.0 mg l−1 each of 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA). A pulse treatment of 0.5 mg l−1 thidiazurone (TDZ) and 0.1 mg l−1 IBA for 5 days was necessary for shoot organogenesis in green compact callus before subculture into 0.5 mg l−1 BA and 0.1 mg l−1 IBA containing medium. Leaf explants of J. curcas, collected from the field, contained endophytic bacterial contamination, which expressed itself after 2–3 subcultures. These bacteria were cultured and identified as Enterobacter ludwigii. After staining, these were found as gram-negative bacteria. Their sensitivity against different antibiotics has been tested by culturing them with different antibiotic stabs for 72 h. Finally, Augmentin® was found as the most effective and suitable antibiotic which not only controlled the bacteria within 2–3 subcultures but also supported the regeneration system and growth of the regenerated shoots and such cultures have been grown for a long-term of over 2 years without any contamination.

복합생균제 첨가가 버섯부산물의 화학적 성분 변화와 발효 저장성에 미치는 영향

This study was conducted to evaluate effects of mixed microbes addition on physico-chemical, fermentative and microbial parameters of sawdust-based spent mushroom substrates(SMS). The SMS was inoculated with mixed microbes(Enterobacter ludwigii, Bacillus cereus, 2 strains of Bacillus subtillis, Saccharomyces cerevisiae and Lactobacillus plantarum) at 1% level(wet basis) and anaerobically fermented during the different periods(up to 8th week). Compared with the SMS before ensiling, the ensiled one had higher CP, NDF and ADF percentages and lower DM and NFC percentages. However, levels of change were very low. The in situ ruminal disappearance of SMS DM and NDF decreased with the ensiling period prolonged. For fermentative parameters, pH reduced and lactic acid contents increased after ensiling, compared with those after ensiling. At 8th week of ensiling, pH increased and lactic acid contents reduced again, compared with those at 4th week of ensiling; however, the silage still showed favorable fermentation status. Lactic acid bacteria counts did not change throughout 8 weeks of ensiling. Counts of total microbes and yeast reduced after 4th week of ensiling period. Counts of lactic acid bacteria and yeast at 8th week of ensiling were in the levels of 108cfu/g. These results indicate that anaerobic fermentation with microbial addition could be an effective way for the long term(8 weeks) storage of the SMS.

Plant growth promoting properties of a strain of Enterobacter ludwigii isolated from Lolium perenne rhizosphere

The capability of native bacterial strains isolated from Lolium perenne rhizosphere to behave as plant growth promoting bacteria and /or biocontrol agents was investigated. One strain (BNM 0357) over 13 isolates from the root tips of L. perenne resulted proved to be nitrogenase positive (ARA test) and an IAA producer. Conventional tests and the API 20E diagnostic kit indicated that BNM 0357 behaves to the Enterobacteriaceae family and to the Enterobacter genus. Molecular identification by 16S rRNA sequence analysis indicated that BNM 0357 had the highest similarity to Enterobacter ludwigii (EN-119). Isolate BNM 0357 had the capability to solubilize calcium triphosphate and to antagonize Fusarium solani mycelial growth and spore germination. Strain BNM 0357 also showed the ability to improve the development of the root system of L. perenne. This study disclosed features of E. ludwigii BNM 0357 that deserve further studies aimed at confirming its putative importance as a PGPR.

버섯부산물 퇴적발효 시 섬유소 분해균 접종이 섬유소 분해성 효소 활력과 면양의 영양소 이용성에 미치는 영향

ABSTRACT This study was conducted to determine effects of cellulolytic microbes inoculation to sawdust-based spent mushroom substrate (SMS) during deepstacking on fermentation parameters, total microbial counts and cellulolytic enzyme activity and to on SMS nutrients utilization by sheep. For sheep metabolism trials, six sheep (ram, average 54.8 kg) were fed a Control diet (70% concentrates, 15% rice straw and 15% SMS with no microbial treatment on a dry basis) and a Treatment diet (the same diet including SMS with a microbial treatment) for 2 trials. Spent mushroom substrates with or without a microbial (4 strains including 1 strain of Enterobacter ludwigii , 1 strain of Bacillus cereus and 2 strains of Bacillus subtillis ) treatment (1% of SMS on wet basis) were deepstacked for 7 days. The internal temperatures in 1.2 M/T of SMS deepstacks reached to 50 ± 5within 7 days of storage. Total microbial counts remarkably decreased (P 0.05) by the microbial treatment. For fibrolytic enzyme activity, CMCase and xylanase activities were decreased (P 0.05) by feeding of microbially treated SMS. Accordingly, these results indicate that cellulolytic microbes inoculation during deepstacking of SMS may improve the bio- utilization of SMS by sheep.(

Description of Enterobacter ludwigii sp. nov., a novel Enterobacter species of clinical relevance

A new species, Enterobacter ludwigii, is presented on the basis of the characteristics of 16 strains, which were isolated from clinical specimens. These bacteria form a distinct genetic cluster in phylogenetic analyses of the population structure of the Enterobacter cloacae complex. As determined by DNA–DNA cross-hybridization experiments in microplates, this genetic cluster can be delineated from the other species of the E. cloacae complex with Δ T m values equal to or above 5 °C with Enterobacter hormaechei being the closest relative. The bacteria are gram-negative, fermentative, motile rods with the general characteristics of the genus Enterobacter and the E. cloacae complex in particular. E. ludwigii can be differentiated from the other Enterobacter species by its growth on myo-inositol and 3-0-methyl- d-glucopyranose. The type strain is EN-119 (=DSM 16688 T=CIP 108491 T).