Characterization and Phylogenetic Analysis of Microbial Surface Active Compound-Producing Bacteria

Microbial surface active compounds (SACs) were isolated from various environmental sources in Thailand. Isolates were screened for SACs production in different carbon sources (crude glycerol, commercial sugar, decanter, glucose, molasses, used palm oil, and used lubricating oil) by using drop-collapsing test and emulsification activity. Molasses produced the highest number of positive results (23 of 25 isolates). Twenty-one isolate strains produced emulsions with xylene, and 15 exhibited high emulsion-stabilizing capacity, maintaining more than 50?% of the original emulsion volume for 24?h, and six isolate strains reduced the growth medium surface tension to 40?mN/m. The phylogenetic position of these 25 isolates was evaluated by 16S rRNA gene sequence analysis. The production of microbial SACs was determined for strains representative of 16 different bacterial genera, in which ten genera (Blastococcus, Erysipelothrix, Humicoccus, Methylophilus, Microlunatus, Nevskia, Pectinatus, Rubrimonas, Selenomonas, and Stenotrophomonas) were firstly reported as SAC-producing strains. Overall, the new SAC-producing strains isolated in this study display promising features for the future development and use in economically efficient industrial-scale biotechnological processes.     

Degradation of bisphenol A by <Emphasis Type="Italic">Bacillus pumilus</Emphasis> isolated from kimchi,a traditionally fermented food

Novel bisphenol A (BPA)-degrading bacterial strains, designated as BP-2CK, BP-21DK, and BP-22DK, were isolated from kimchi, a traditionally fermented food. These isolates were identified as Bacillus pumilus and efficiently degraded BPA in a medium supplemented with nutrients such as peptone, beef extract, and yeast extract. Strains BP-2CK, BP-21DK, and BP-22DK successfully degraded 25, 25, and 50 ppm of BPA, respectively, and all strains exhibited BPA-degrading activity in the presence of 10% NaCl. Accumulation of the metabolites including 4-hydroxyacetophenone, one of the intermediates produced by the other BPA-degrading bacteria, was not observed in BPA degradation by the isolated strains. These results indicate that the isolated food-derived bacteria are applicable for the construction of efficient and safer systems for the removal of BPA.     

Molecular Characterization of Bacterial Isolates from Soil Samples and Evaluation of their Antibacterial Potential against MDRS

Some soil microbes, with their diverse inhabitance, biologically active metabolites, and endospore formation, gave them characteristic predominance and recognition among other microbial communities. The present study collected ten soil samples from green land, agricultural and marshy soil sites of Khyber Pakhtunkhwa, Pakistan. After culturing on described media, the bacterial isolates were identified through phenotypic, biochemical and phylogenetic analysis. Our phylogenetic analysis revealed three bacterial isolates, A6S7, A1S6, and A1S10, showing 99% nucleotides sequence similarity with Brevibacillus formosus, Bacillus Subtilis and Paenibacillus dendritiformis. The crude extract was prepared from bacterial isolates to assess the anti-bacterial potential against various targeted multidrug-resistant strains (MDRS), including Acinetobacter baumannii (ATCC 19606), Methicillin-resistant Staphylococcus aureus (MRSA) (BAA-1683), Klebsiella pneumoniae (ATCC 13883), Pseudomonas aeruginosa (BAA-2108), Staphylococcus aureus (ATCC 292013), Escherichia coli (ATCC25922) and Salmonella typhi (ATCC 14028). Our analysis revealed that all bacterial extracts possess activity against Gram-negative and Gram-positive bacteria at a concentration of 5 mg/mL, efficiently restricting the growth of E. coli compared with positive control ciprofloxacin. The study concluded that the identified species have the potential to produce antimicrobial compounds which can be used to control different microbial infections, especially MDRS. Moreover, the analysis of the bacterial extracts through GC-MS indicated the presence of different antimicrobial compounds such as propanoic acid, oxalic acid, phenol and hexadecanoic acid.     

Biochemical Basis of Mercury Remediation and Bioaccumulation by Enterobacter sp. EMB21

The aims of this study were to isolate metal bioaccumulating bacterial strains and to study their applications in removal of environmental problematic heavy metals like mercury. Five bacterial strains belonging to genera Enterobacter, Bacillus, and Pseudomonas were isolated from oil-spilled soil. Among these, one of the strains Enterobacter sp. EMB21 showed mercury bioaccumulation inside the cells simultaneous to its bioremediation. The bioaccumulation of remediated mercury was confirmed by transmission electron microscopy and energy dispersive X-ray. The mercury-resistant loci in the Enterobacter sp. EMB21 cells were plasmid-mediated as confirmed by transformation of mercury-sensitive Escherichia coli DH5α by Enterobacter sp. EMB21 plasmid. Effect of different culture parameters viz-a-viz inoculum size, pH, carbon, and nitrogen source revealed that alkaline pH and presence of dextrose and yeast extract favored better remediation. The results indicated the usefulness of Enterobacter sp. EMB21 for the effective remediation of mercury in bioaccumulated form. The Enterobacter sp. EMB21 seems promising for heavy metal remediation wherein the remediated metal can be trapped inside the cells. The process can further be developed for the synthesis of valuable high-end functional alloy, nanoparticles, or metal conjugates from the metal being remediated.     

Chemical composition and antimicrobial activity of Satureja kitaibelii essential oil against pathogenic microbial strains

Plant species Satureja kitaibelii Wierzb. ex Heuff. is used as a spice and as a natural preservative for food and herbal tea, owing to its characteristic scent and flavor as well as high antimicrobial activity. In the present study, the antimicrobial activity of isolated essential oil of S. kitaibelii was tested against a panel of 30 pathogenic microorganisms (foodborne microbes, selected multiresistant bacterial isolates from the patient wounds and dermatophyte isolates). Limonene (15.54%), p-cymene (9.99%), and borneol (8.91%) appeared as the main components in 44 identified compounds representing 98.44% of the oil. Essential oil of S. kitaibelii showed significant activity against a wide spectrum of foodborne microbes (MIC=0.18-25.5 microg mL(-1)) and multiresistant bacterial isolates (MIC=6.25-50.0 microg mL(-1)), as well as against dermatophyte strains (MIC=12.5-50.0 microg mL(-1)). These results demonstrate that S. kitaibelii essential oil could be used as a natural potential antimicrobial agent against pathogenic strains in the treatment of foodborne disease, wound and skin infections.     

Poly-β-hydroxybutyrate and Exopolysaccharide Biosynthesis by Bacterial Isolates from Pigeonpea [<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) Millsp] Root Nodules

The bacterial strains that are able to produce biopolymers that are applied in industrial sectors present a source of renewable resources. Some microorganisms are already applied at several industrial sectors, but the prospecting of new microbes must bring microorganisms that are feasible to produce interesting biopolymers more efficiently and in cheaper conditions. Among the biopolymers applied industrially, polyhydroxybutyrate (PHB) and exopolysaccharides (EPS) stand out because of its applications, mainly in biodegradable plastic production and in food industry, respectively. In this context, the capacity of bacteria isolated from pigeonpea root nodules to produce EPS and PHB was evaluated, as well as the cultural characterization of these isolates. Among the 38 isolates evaluated, the majority presented fast growth and ability to acidify the culture media. Regarding the biopolymer production, five isolates produced more than 10 mg PHB per liter of culture medium. Six EPS producing bacteria achieved more than 200 mg EPS per liter of culture medium. Evaluating different carbon sources, the PHB productivity of the isolate 24.6b reached 69% of cell dry weight when cultured with starch as sole carbon source, and the isolate 8.1c synthesized 53% PHB in dry cell biomass and more than 1.3 g L⁻¹ of EPS when grown using xylose as sole carbon source.     

Isolation and Characterization of Putative Endophytic Bacteria Antagonistic to Phoma tracheiphila and Verticillium albo-atrum

A collection of 200 bacterial isolates recovered from citrus plants (Citrus limon, Citrus sinensis, and Citrus reticulata), Medicago truncatula and Laurus nobilis, was established. In vitro screening indicated that 28 isolates exhibited an inhibitory activity against the vascular pathogens Phoma tracheiphila and Verticillium albo-atrum. Isolates were screened according to their hydrolytic activities, plant growth-promoting bacteria (PGPB) abilities, as well as for the presence of nonribosomal peptide synthetase (NRPS) genes responsible of the lipopeptide biosynthesis. The results were positive for 16 isolates which exhibited at least two PGPB activities and a single NRPS gene. Genetic diversity of the selected isolates was studied using random amplified polymorphic DNA (RAPD) and repetitive element PCR (REP) tools that showed clustering of strains into three major groups (I, II, and III) (i, ii, and iii), respectively. Clustering was further confirmed by the 16S rDNA sequencing that assigned nine isolates to Bacillus velezensis, four isolates to Bacillus methyltrophicus, one isolate to Bacillus amyloliquefaciens, and two isolates to Bacillus mojavensis. Organ-bacterial genotype interaction as well as positive correlation with NRPS genes are discussed.     

Capillary electrophoresis of microbial aggregates

Uncontrolled aggregation of bacterial cells is a significant disadvantage of electrophoretic separations. Various aspects of the electrophoretic behavior of different strains of Gram‐positive Bacillus cereus, Bacillus subtilis, Sarcina lutea, Staphylococcus aureus(1), and Micrococcus luteus bacteria and Gram‐negative Escherichia coli bacteria were investigated in this study. Our findings indicate that bacteria can be rapidly analyzed by CZE with surface charge modification by calcium ions (Ca²⁺). Bound Ca²⁺ ions increase zeta potential to more than 2.0 mV and significantly reduce repulsive forces. Under the above conditions, bacterial cells create compact aggregates, and fewer high‐intensity signals are observed in electropherograms. The above can be attributed to the bridging effect of Ca²⁺ between bacterial cells. CE was performed to analyze bacterial aggregates in an isotachophoretic mode. A single peak was observed in the electropherogram.     

Degradation of PCB by Bacteria Isolated from Long-Time Contaminated Soil

Abstract

Four bacterial isolates belonging to the genera Pseudomonas and Alcaligenes were obtained by the enrichment method, using biphenyl as the sole carbon source, from the soil, which underwent long-time contamination with technical mixtures of PCB. Kinetics of PCB degradation by individual isolates was measured using n-hexane extraction of the cultivation media in proper time intervals and analysed by congener specific gas chromatography with electron capture detection. All isolates exhibit interesting biodegradative potential. Specific degradation of individual congeners with respect to the number and position of chlorine substituents is discussed. The influence of glucose, biphenyl and 3-chlorobenzoic acid on the PCB degradation has been assessed.     

Adhesion of waste water bacteria to reverse osmosis membranes

A stirred cell was used to study initial adhesion of three sewage bacteria belonging to the genus Pseudomonas to the three reverse osmosis (RO) membranes BW30, PVD and CAB2, and the nanofiltration membrane NF45. Membranes were immersed in suspensions containing 10⁸ bacteria/ml for 10 min. All three strains were capable of rapidly colonising the four membranes, but to different extents. It was found that bacteria would sometimes aggregate upon adhering to particular RO membranes. The effects of solution ionic strength and pH, and conditioning of membranes (by prior exposure to filtrates of treated and untreated sewage) on the number of adherent bacteria were investigated. Minimal bacterial attachment occurred in a very low ionic strength milieu (deionised water). Salt concentrations corresponding to waste water and to twice that concentration resulted in significantly higher but statistically similar numbers of attached microbes. Adhesion of the three isolates was not affected by pH in the range of 4–8. The number of bacteria attaching to the membranes could be increased or reduced by conditioning films of sewage origin, conditioning films could also trigger or inhibit aggregation of adherent cells. Some surface properties of the membranes (roughness, hydrophobicity) and bacterial cells (electrophoretic mobility, functional groups by affinity chromatography) were also investigated.     

Composition and antimicrobial activity of Seseli globiferum essential oil

The essential oil from aerial parts of Seseli globiferum Vis. obtained by hydrodistillation with Clevenger-type apparatus was analyzed by GC-MS. Twenty-eight compounds were identified, representing 99.4% of the total oil. The main components of the oil were sabinene (38.0%), alpha-pinene (21.2%) and beta-phellandrene (13.5%). The microbial growth inhibitory properties of the isolated essential oil were determined using the broth microdilution method against seven bacterial species: Salmonella typhimurium (ATCC 13311), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Enterobacter cloacae (clinical isolates), Bacillus cereus (clinical isolates), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Micrococcus flavus (ATCC 10240) and three fungal species: Aspergillus niger (ATCC 6275), Aspergillus versicolor (ATCC 11730), Trichoderma viride (IAM 5061) and Penicillium funiculosum (ATCC 36839). The essential oil showed activity against bacteria P. aeruginosa, followed by M flavus, L. monocytigenes and E. coli, and all investigated fungal species.     

Rapid serotyping of enteropathogenic Yersinia Enterocolitica strains by co-agglutination

A rapid co-agglutination test using monospecific antisera was developed for the serological typing of enteropathogenic strains of Yersinia enterocolitica. A total of 70 bacterial strains (17 reference strains and 53 clinical isolates) were examined. Absorption of immune sera against srotypes O:3, O:8 and O:9 with their heterologous antigens (S-LPS) was necessary to avoid the appearance of different cross-reactions, as observed by co-agglutination. The proteins present in the S-LPS preparations obtained from each serotype seemed to be responsible for such cross-reactions. Results obtained with a total of 57 clinical isolates belonging to other members of the family Enterobacteriaceae indicated a high specificity of the assay.     

Cloning,Sequence Analysis and Three-dimensional Structure Prediction of DNA Pol I from Thermophilic <Emphasis Type="Italic">Geobacillus</Emphasis> sp. <Emphasis Type="Italic">MKK</Emphasis> Isolated from an Iranian Hot Spring

Molecular phylogenetic analysis of a novel thermophilic eubacterium isolated from an Iranian hot spring using 16S rDNA sequence showed that the new isolate belongs to genera Geobacillus. DNA pol I gene from this isolate was amplified, cloned, sequenced, and the three-dimensional (3D) structure of deduced amino acid sequence was predicted. Sequence analysis revealed the gene is 2,631 bp long, encodes a protein of 876 amino acids with a calculated molecular mass of 99 kDa, and belongs to family A DNA polymerases. Comparison of 3′–5′exonuclease domain of Klenow fragment (KF) with corresponding region of newly identified DNA pol I (MF), the large fragment of Bacillus stearothermophilus DNA pol I (BF) and Klentaq1, revealed not only deletions in three regions compared to KF, but that three of the four critical metal-binding residues in KF (Asp355, Glu357, Asp424, and Asp501) are altered in MF as well. Predicted 3D structure and sequence alignments between MF and BF showed that all critical residues in the polymerase active site are conserved.     

A scalable microfluidic chip for bacterial suspension culture

Microfluidic systems could, in principle, enable high-throughput breeding and screening of microbial strains for industrial applications, but parallel and scalable culture and detection chips are needed before complete microbial selection systems can be integrated and tested. Here we demonstrate a scalable multi-channel chip that is capable of bacterial suspension culture. The key invention is a multi-layered chip design, which enables a single set of control channels to function as serial peristaltic pumps to drive parallel culture chamber loops. Such design leads to scalability of the culture chip. We demonstrate that E. coli growth in the chip is equivalent or superior to conventional suspension culture on shaking beds. The chip could also be used for suspension culture of other microbes such as Bacillus subtilis, Pseudomonas stutzeri, and Zymomonas mobilis, indicating its general applicability for bacterial suspension culture.     

A Comprehensive Analysis Using Colorimetry,Liquid Chromatography-Tandem Mass Spectrometry and Bioassays for the Assessment of Indole Related Compounds Produced by Endophytes of Selected Wheat Cultivars

Liquid chromatography–tandem mass spectrometry (LC–MS/MS), colorimetry, and bioassays were employed for the evaluation of the ability of endophytic bacterial strains to synthesize indole-related compounds (IRCs) and in particular indole-3-acetic acid (IAA). A total of 54 endophytic strains belonging to seven bacterial genera isolated from tissues of common and spelt wheat cultivars were studied. The endophytic bacteria isolated from different tissues of the tested wheat types were capable of IRCs production, including IAA, which constituted from 1.75% to 52.68% of all IRCs, in in vitro conditions via the tryptophan dependent pathway. The selected post-culture medium was also examined using a plant bioassay. Substantial growth of wheat coleoptile segments treated with the bacterial post-culture medium was observed in several cases. Our data suggest that the studied endophytic bacteria produce auxin-type compounds to support plant development. Summarizing, our approach to use three complementary methods for estimation of IRCs in different endophytic strains provides a comprehensive picture of their effect on wheat growth.     

Degradation of abiotically aged LDPE films containing pro-oxidant by bacterial consortium

The degradation of abiotically aged low density polyethylene (LDPE) films containing trace quantities of a representative pro-oxidant (cobalt stearate) was investigated in the presence of well defined enriched microbial strains namely, Bacillus pumilus, Bacillus halodenitrificans and Bacillus cereus in Basal salt medium. The films were initially subjected to an abiotic treatment comprising UV-B irradiation, and subsequently inoculated with the bacterial strains. The degradation in the polymeric chain was monitored by changes in the mechanical, morphological, structural and thermal properties. The abiotic treatment led to the formation of extractable oxygenated compounds as well as unoxidised low molecular weight hydrocarbons, which was confirmed by GC-MS studies. These were utilized by the bacterial consortium in the subsequent biotic phase and led to a mass loss of the polymer (8.4 ± 1.37%), which was also accompanied by an increase in the bacterial count. A decrease in the surface tension of the cell free medium was observed, which indicates that the bacterial consortium produced extracellular surface active molecules in order to enhance the bioavailability of the polymeric fixed carbon. The spectroscopic investigations reveal that the bacteria preferentially consume the oxygenated products leading to a decrease in the Carbonyl Index (CI), which in turn leads to an increase in the initial decomposition temperature as observed in the TGA traces. The morphological investigations reveal a biofilm formation on the surface, which was found to be scattered in certain regions and not uniform on the polymeric surface.     

Another Bacillus sphaericus serotype harbouring strains very toxic to mosquito larvae: Serotype H6

Ten isolates of Bacillus sphaericus from Ghana, very toxic to mosquito larvae, have been identified as belonging to serotype H6. These isolates can be represented by the head-group strain IAB59. They form crystals at the sporulation stage. Their larvicidal effect on Culex pipiens and Anopheles stephensi larvae is as high as that of the most toxic strains already known, e.g. 1593 and 2362 (serotype H5a,5b) and 2297 (serotype H25). Spore-crystal extracts of all these strains contain a 43-Kd polypeptide immunologically related to the 43-Kd polypeptide from strain 2362 described by other authors.     

Isolation and characterization of a novel feather-degrading bacterial strain

Feather waste, generated in large quantities as a byproduct of commercial poultry processing, is almost pure keratin, which is not easily degradable by common proteolytic enzymes. Feather-degrading bacteria were isolated from a Brazilian poultry industrial waste. Among these isolates, a strain identified as kr2 was the best feather-degrading organism when grown on basal medium containing 10 g/L of native feather as a source of energy, carbon, and nitrogen. The isolate was characterized according to morphological characteristics and biochemical tests belonging to the Vibrionaceae family. Keratinolytic activity of this isolate was monitored throughout the cultivation of the bacterium on raw feather at different temperatures. The optimum temperature for growth was about 30°C, at which maximum enzyme and soluble protein production were achieved. The enzyme had a pH and temperature optima of 8.0 and 55°C, respectively.     

芽胞杆菌全细胞热裂解气相色谱聚类分析

通过热裂解气相色谱对１４ 个芽胞杆菌模式菌株全细胞进行分析, 确定芽胞杆菌不同属间特征性谱峰;利用ＳＴＡＴＩＳＴＩＣＡ 系统对热裂解气相色谱图中３０ 个主要谱峰进行聚类分析, 绘制出的树状谱清楚地表明各菌株间的亲缘关系。     

Studies on drug metabolism by fungi colonizing decomposing human cadavers. Part I: DNA sequence-based identification of fungi isolated from postmortem material

Cadavers can be colonized by a wide variety of bacteria and fungi. Some of these microbes could change the concentration or the metabolic pattern of drugs present in postmortem samples. The purpose of this study was to identify fungi from human postmortem material and to further assess their potential role in the metabolism of drugs. Aliquots of 252 postmortem samples (heart blood, liver, kidney, and lung) taken from 105 moderately to severely decomposed bodies were streaked on Sabouraud agar for isolation of fungal species. One part of the samples was worked up immediately after autopsy (group I). The second part had previously been stored at ?20 °C for at least 1 year (group II). Identification of the isolates was achieved morphologically by microscopy and molecularly by polymerase chain reaction amplification and sequencing of markers allowing species identification of the respective genera. Depending on the genus, different gene fragments were used: calmodulin for Aspergillus, β-tubulin for Penicillium, translation elongation factor 1α for Fusarium, and the internal transcribed spacer region of the ribosomal DNA for all remaining genera. A total of 156 fungal strains were isolated from 62 % of the postmortem materials. By using these primers, 98 % of the isolates could be identified to the species level. The most common genera were Candida (60.0 %—six species), Penicillium (10.3 %—two species), Rhodotorula (7.1 %—one species), Mucor (6.4 %—four species), Aspergillus (3.2 %—four species), Trichosporon (3.2 %—one species), and Geotrichum (3.2 %—one species). Group I samples contained 53 % more fungal species than stored samples suggesting some fungi did not survive the freezing process. The isolated fungi might be characteristic for decomposed bodies. The proposed methodology proved to be appropriate for the identification of fungi in this type of material.