Polyphasic characterization and genomic insights into Nocardioides turkmenicus sp. nov. isolated from a desert soil.

Strain KC13T, a novel desert-adapted, non-motile, Gram-stain-positive, rod-shaped, aerobic bacterium, was isolated from a soil sample collected from the Karakum Desert, Turkmenistan and characterised by a polyphasic approach. Phylogenetic analysis based on 16S rRNA sequences revealed that strain KC13T was a member of the genus Nocardioides, and formed a distinct cluster with Nocardioides luteus DSM 43366T (99.3% sequence identity), Nocardioides albus DSM 43109T (98.9%), Nocardioides panzhihuensis DSM 26487T (98.3%) and Nocardioides albertanoniae DSM 25218T (97.9%). The orthologous average nucleotide identity and digital DNA-DNA hybridization values were in the range of 85.8-91.0% and 30.2-35.9%, respectively, with the type strains of closely related species. The genome size of strain KC13T was 5.3 Mb with a DNA G + C content of 69.7%. Comprehensive genome analyses showed that strain KC13T, unlike its close relatives, had many genes associated with environmental adaptation. Strain KC13T was found to have chemotaxonomic and phenotypic characteristics of members of the genus Nocardioides and some differences from phylogenetic neighbours. Based on the chemotaxonomic, genomic, phenotypic and phylogenetic data, strain KC13T represents a novel species of the genus Nocardioides, for which the name Nocardioides turkmenicus sp. nov. is proposed, and the type strain is KC13T (= JCM 33525T = CGMCC 4.7619T).

In vitro genotoxic and antigenotoxic effects of an exopolysaccharide isolated from Lactobacillus salivarius KC27L.

Exopolysaccharide isolated from Lactobacillus salivarius (new genus name Ligilactobacillus) KC27L strain (EPSKC27L) exhibits antioxidant properties with 1,1-diphenyl-2-picrylhydrazase (DPPH) radical and superoxide anion radical (O2-.) scavenging effect and iron ion (Fe2+) chelating activity. This study aimed to investigate the in vitro genotoxic effects of EPSKC27L alone (12.50, 25.00, 50.00, and 100.00 µg/mL) and its antigenotoxic activity against DNA damage induced by mitomycin-C (MMC; 0.20 µg/mL), methyl methanesulfonate (MMS; 5.00 µg/mL), and hydrogen peroxide (H2O2; 100 µM). For this purpose, chromosome aberration (CA), sister chromatid exchange (SCE), micronucleus (MN), and comet assays were performed in human peripheral lymphocytes. In addition, the structure of EPSKC27L was investigated in the scanning electron microscope (SEM). EPSKC27L alone did not cause a significant genotoxic effect in CA, SCE, MN, and comet tests. EPSKC27L significantly decreased the frequency of CA, SCE, and MN induced by MMC and MMS. EPSKC27L also significantly reduced DNA damage induced by H2O2. This study showed that the EPSKC27L alone has no genotoxic risk at these concentrations and shows antigenotoxic activity against MMC, MMS, and H2O2. Consequently, EPSKC27L was found to exhibit chemopreventive activity against genotoxic agents. This effect is believed to be due to the antioxidant properties of EPSKC27L.

Escherichia coli Enumeration in a Capillary-Driven Microfluidic Chip with SERS.

Pathogen detection is still a challenging issue for public health, especially in food products. A selective preconcentration step is also necessary if the target pathogen concentration is very low or if the sample volume is limited in the analysis. Plate counting (24-48 h) methods should be replaced by novel biosensor systems as an alternative reliable pathogen detection technique. The usage of a capillary-driven microfluidic chip is an alternative method for pathogen detection, with the combination of surface-enhanced Raman scattering (SERS) measurements. Here, we constructed microchambers with capillary microchannels to provide nanoparticle-pathogen transportation from one chamber to the other. Escherichia coli (E. coli) was selected as a model pathogen and specific antibody-modified magnetic nanoparticles (MNPs) as a capture probe in a complex milk matrix. MNPs that captured E. coli were transferred in a capillary-driven microfluidic chip consisting of four chambers, and 4-aminothiophenol (4-ATP)-labelled gold nanorods (Au NRs) were used as the Raman probe in the capillary-driven microfluidic chip. The MNPs provided immunomagnetic (IMS) separation and preconcentration of analytes from the sample matrix and then, 4-ATP-labelled Au NRs provided an SERS response by forming sandwich immunoassay structures in the last chamber of the capillary-driven microfluidic chip. The developed SERS-based method could detect 101-107 cfu/mL of E. coli with the total analysis time of less than 60 min. Selectivity of the developed method was also tested by using Salmonella enteritidis (S. enteritidis) and Staphylococcus aureus (S. aureus) as analytes, and very weak signals were observed.

Influence of graphene oxide on the toxicity of polystyrene nanoplastics to the marine microalgae Picochlorum sp.

Graphene oxide (GO) features distinctive physical and chemical characteristics; therefore, it has been intensively investigated in environmental remediation as a promising material for clean-up of soil contamination and water purification and used as immobilization material. Plastic is a widespread pollutant, and its breakdown products such as nanoplastics (NPs) should be evaluated for potential harmful effects. This study is aimed to evaluate the influence of GO on the toxicity of polystyrene (PS) NPs to the marine microalgae Picochlorum sp. over a period of 4 weeks. The capability of GO to reduce the toxic effects of PS NPs was assessed through investigating exposure sequence of GO in the presence of 20 nm diameter-sized polystyrene NPs. This was accomplished through five test groups: microalgae pre-exposed to GO prior to incubation with PS NPs, microalgae post-exposed to GO after incubation with PS NPs, microalgae simultaneously exposed to GO and PS NPs, and individual exposure of microalgae to either GO or PS NPs. Cytotoxicity assay results demonstrated that microalgae pre-exposed to GO prior to incubation with PS NPs showed an increased viability and chlorophyll a content. The pre-exposure to GO has reduced the growth inhibition rate (IR) from 50%, for microalgae simultaneously exposed to GO and PS NPs, to 26%, for microalgae pre-exposed to GO. Moreover, the lowest level of reactive oxygen species (ROS) was recorded for microalgae exposed to GO only and microalgae pre-exposed to GO. Fourier-transform infrared (FTIR) analysis, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) observations revealed some morphological changes of both algae and their extracellular polymeric substances (EPS) upon GO and PS NPs exposure combinations. The sequence of GO exposure to aquatic microorganisms might affect the level of harm caused by the PS NPs. Therefore, application of GO as part of an immobilization material and in the removal of pollutants from water should be carefully investigated using different pollutants and aquatic organisms.

Saccharopolyspora soli sp. nov., isolated from Northern Cyprus soil.

A novel Gram-stain positive, aerobic, non-motile actinobacterium, designated strain K220T, was isolated from soil collected from Cape Andreas (Zafer Burnu), Northern Cyprus, and subjected to a polyphasic taxonomic approach. The organism was shown to have phylogenetic, chemotaxonomic, cultural and morphological properties consistent with its classification in the genus Saccharopolyspora. 16S rRNA gene sequence analysis of strain K220T showed that it is closely related to the type strains of Saccharopolyspora maritima 3SS5-12 T, Saccharopolyspora kobensis JCM 9109 T and Saccharopolyspora hirsuta ATCC 27875 T with 97.6, 97.5 and 97.0% sequence similarity, respectively. In silico DNA-DNA hybridization and average nucleotide identity values between strain K220T and type strains of the genus Saccharopolyspora with publicly available genomes were 22.1-31.2% and 76.0-83.16%, respectively. The DNA G + C content of strain K220T was 68.3 mol%. The genome of strain K220T has genes associated with 24 biosynthetic gene clusters. The strain contained MK-9(H4) and iso-C16: 0 as the predominant respiratory quinone and fatty acid, respectively. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine. Based on evidence collected from the genotypic, phenotypic and phylogenetic analyses, strain K220T is considered to represent a novel species in the genus Saccharopolyspora, for which the name Saccharopolyspora soli sp. nov. is proposed. The type strain is K220T (= JCM 33912T = KCTC 49395T).

Actinomadura soli sp. nov., isolated from the top soil layer on basaltic material in Turkey.

An actinobacterium, designated 14C53T, was isolated from a soil sample on basaltic material from Samsun, Turkey. The growth ranges for NaCl concentration and pH of strain 14C53T were quite limited and the growth temperature range of the strain was 20-37 °C, with an optimum at 28 °C. Phylogenetic analysis of 16S rRNA gene sequences revealed that strain 14C53T was most closely related to Actinomadura geliboluensis A8036T (98.5 % similarity value), but in the phylogenetic tree, it formed a clade with Actinomadura alkaliterrae D310AT. The genome tree revealed a close relationship between the strain and Actinomadura pelletieri DSM 43383T. However, the digital DNA-DNA hybridization and average nucleotide identity values between strain 14C53T with Actinomadura geliboluensis A8036T and Actinomadura pelletieri DSM 43383T were 28.6-30.2 % and 84.3-85.5 %, respectively, and comparative analyses based on the genome sequences demonstrated that it represents a novel species of the genus Actinomadura. The genome size of strain 14C53T was approximately 9.0 Mb and the genomic DNA G+C content of the strain was 71.3 mol%. The major cellular fatty acids of strain 14C53T were C16 : 0 and iso-C16 : 0. Strain 14C53T contained meso-diaminopimelic acid as the diamino acid in the cell-wall peptidoglycan. The predominant menaquinones were MK-9(H8) and MK-9(H6). Based on evidence collected from the phenotypic, genotypic and phylogenetic analyses, a novel species Actinomadura soli sp. nov. is proposed, with 14C53T (=DSM 104447T=KCTC 39878T) as the type strain.

Saccharopolyspora karakumensis sp. nov., Saccharopolyspora elongata sp. nov., Saccharopolyspora aridisoli sp. nov., Saccharopolyspora terrae sp. nov. and their biotechnological potential revealed by genome analysis.

Exploration of unexplored habitats for novel actinobacteria with high bioactivity potential holds great promise in the search for novel entities. During the course of isolation of actinobacteria from desert soils, four actinobacteria, designated as 5K548T, 7K502T, 16K309T and 16K404T, were isolated from the Karakum Desert and their bioactivity potential as well as taxonomic provenances were revealed by comprehensive genome analyses. Pairwise sequence analyses of the 16S rRNA genes indicated that the four strains are representatives of putatively novel taxa within the prolific actinobacterial genus Saccharopolyspora. The strains have typical chemotaxonomic characteristics of the genus Saccharopolyspora by having meso-diaminopimelic acid as diagnostic diaminoacid, arabinose, galactose and ribose as whole-cell sugars. Consistent with this assignment, all of the isolates contained phosphatidylcholine in their polar lipid profiles and MK-9(H4) as the predominant menaquinone. The sizes of the genomes of the isolates ranged from 6.0 to 10.2 Mb and the associated G + C contents from 69.6 to 69.7 %. Polyphasic characterizations including determination of overall genome relatedness indices revealed that the strains are representatives of four novel species in the genus Saccharopolyspora. Consequently, isolates 5K548T, 7K502T, 16K404T and 16K309T are proposed as novel Saccharopolyspora species for which the names of Saccharopolyspora karakumensis sp. nov., Saccharopolyspora elongata sp. nov., Saccharopolyspora aridisoli sp. nov. and Saccharopolyspora terrae sp. nov. are proposed, respectively. Comprehensive genome analysis for biosynthetic gene clusters showed that the strains have high potential for novel secondary metabolites. Moreover, the strains harbour many antimicrobial resistance genes providing more evidence for their potentiality for bioactive metabolites.

Comprehensive genome analysis of a novel actinobacterium with high potential for biotechnological applications, Nonomuraea aridisoli sp. nov., isolated from desert soil.

During a study to isolate such actinobacteria with unique metabolic potential, a novel actinobacterium, designated KC333T, was isolated from a soil sample collected from the Karakum Desert, Turkmenistan. The taxonomic position of the strain was investigated using a polyphasic approach. Phylogenetic analysis of the 16S rRNA gene sequence showed that the strain was most closely related to Nonomuraea terrae CH32T (99.0% sequence similarity), Nonomuraea maritima FXJ7.203 T (98.9%), Nonomuraea candida HMC10T (98.7%) and Nonomuraea gerenzanensis ATCC 39727 T (98.6%), and is therefore considered to represent a member of the genus Nonomuraea. However, the average nucleotide identity and digital DNA-DNA hybridization based on whole-genome sequences between strain KC333T and close relatives demonstrated that it represents a novel species of the genus Nonomuraea. The major cellular fatty acids of strain KC333T were iso-C16: 0, C17:0 10-methyl and iso-C16: 0 2OH. Strain KC333T contained meso-diaminopimelic, mannose, madurose and ribose in the cell-wall peptidoglycan. The predominant menaquinones were MK-9(H4) and MK-9(H6). The genome size of strain KC333T is approximately 9.86 Mb, and the genomic DNA G + C content of the strain is 71.3%. In addition to the polyphasic characterisation, comprehensive genome analysis for gene clusters encoding carbohydrate-active enzymes and bioactive secondary metabolites as well as CRISPR-associated sequences revealed the high biotechnological potential of the strain. Based on evidence collected from the genotypic, phenotypic, and phylogenetic analyses, a novel species, Nonomuraea aridisoli sp. nov. is proposed with KC333T (= DSM 107062 T = JCM 32584 T = KCTC 49111 T) as the type strain.

Multiplex enumeration of Escherichia coli and Salmonella enteritidis in a passive capillary microfluidic chip.

Multiplex detection and quantification of bacteria in water by using portable devices are particularly essential in low and middle-income countries where access to clean drinking water is limited. Addressing this crucial problem, we report a highly sensitive immunoassay sensor system utilizing the fluorescence technique with magnetic nanoparticles (MNPs) to separate target bacteria and two different types of quantum dots (CdTe and Ni doped CdTe QDs) incorporated into a passive microfluidic chip to transport and to form sandwich complexes for the detection of two target bacteria, namely Escherichia coli (E. coli) and Salmonella enteritidis (S. enteritidis) in less than 60 min. The assay is carried out on a capillary driven microfluidic chip that can be operated by merely pipetting the samples and reagents, and fluorescence measurements are done by using a handheld fluorescence spectrophotometer, which renders the system portable. The linear range of the method was found to be 101 to 105 cfu mL-1 for both E. coli and S. enteritidis. The limit of detection (LOD) was calculated to be 5 and 3 cfu mL-1 for E. coli and S. enteritidis, respectively. The selectivity of the method was examined by testing Enterobacter dissolvens (E. dissolvens) and Staphylococcus aureus (S. aureus) samples, and no significant interference was observed. The method was also demonstrated to detect bacteria in tap water and lake water samples spiked with target bacteria.

Investigation of the toxic effects of different polystyrene micro-and nanoplastics on microalgae Chlorella vulgaris by analysis of cell viability, pigment content, oxidative stress and ultrastructural changes.

Plastics of different sizes (micro- and nano-sized) are often identified in aquatic environments. Nevertheless, their influence on marine organisms has not been widely investigated. In this study, the responses of the microalga Chlorella vulgaris to micro- and nanoplastics exposure were examined using long term toxicity test. The plastics tested were carboxyl-functionalized and non-functionalized polystyrene of 20, 50 and 500 nm in diameter. A reduction in algal cell viability and chlorophyll a concentration has been observed after exposure to the small sizes (20 and 50 nm) of plastics. Lactate dehydrogenase activity and reactive oxygen species concentration/production were significantly higher after exposure to the 20 nm nanoplastics than that of control confirming the stress condition. Fourier transform infrared (FTIR) spectroscopy analysis proved the attachment of nanoplastics to microalgae and rearrangement of extracellular polymeric substances. The cellular stress appeared as increased cell size, deformed cell wall and increased volume of starch grains.

Streptomyces boluensis sp. nov., isolated from lake sediment.

A novel Gram-stain positive, aerobic, non-motile actinobacterium, designated strain YC537T, was isolated from lake sediment collected from Yenicaga Lake, Bolu, Turkey, and subjected to a polyphasic taxonomic approach. The organism had phylogenetic, chemotaxonomic, cultural and morphological properties consistent with its classification in the genus Streptomyces. 16S rRNA gene sequence analysis of strain YC537T showed that it is closely related to the type strain of Streptomyces ziwulingensis F22T (97.9% sequence similarity), Streptomyces tauricus JCM 4837 T (97.7%) and Streptomyces beijiangensis NBRC 100044 T (97.6%). The cell wall of the strain contained LL-diaminopimelic acid and the cell-wall sugars were glucose, galactose and ribose. The major phospholipids of strain YC537T were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The predominant menaquinones were identified as MK-9(H6) and MK-9(H8). The major cellular fatty acids were iso-C16:0, iso-C14:0, anteiso-C15:0 and iso-C15:0. Consequently, strain YC537T is considered to represent a novel species in the genus Streptomyces, for which the name Streptomyces boluensis sp. nov. is proposed. The type strain is YC537T (= KCTC 39750 T = DSM 102303 T).

Nonomuraea basaltis sp. nov., a siderophore-producing actinobacteria isolated from surface soil of basaltic parent material.

A Gram-stain-positive, aerobic, spore-forming actinobacterial strain, designated 160415T, was isolated from a surface soil sample, which was formed on basaltic parent material, collected from Samsun, Turkey. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 160415T clustered closely with species of the genus Nonomuraea, and showed the highest sequence similarity to Nonomuraea zeae NEAU-ND5T, Nonomuraea candida HMC10T and Nonomuraea turkmeniaca DSM 43926T with 99.1%, 98.9% and 98.7%, respectively. Chemotaxonomic properties including major menaquinones, diaminopimelic acid, sugar and phospholipid profiles also confirmed the affiliation of the strain to the genus Nonomuraea. The DNA G+C content of strain 160415T was 69.6 mol%. DNA-DNA hybridization and average nucleotide identity values between the strain and closely related type strains were less than the recommended cut-off values. On the basis of phylogenetic relationships, genotypic and phenotypic characterizations, strain 160415T represents a novel species of the genus Nonomuraea, for which the name Nonomuraea basaltis sp. nov. is proposed. The type strain is 160415T (= KCTC 39875T = DSM 104309T).

Streptomyces cahuitamycinicus sp. nov., isolated from desert soil and reclassification of Streptomyces galilaeus as a later heterotypic synonym of Streptomyces bobili.

A novel actinobacterial strain, designated 13K301T, was isolated from a soil sample collected from the Karakum Desert, Turkmenistan. The taxonomic position of strain 13K301T was revealed by using a polyphasic approach. On the basis of 16S rRNA gene sequence analysis, strain 13K301T belongs to the genus Streptomyces and had highest sequence similarity to 'Streptomyces qaidamensis' S10T (99.2 %), Streptomyces flavovariabilis NRRL B-16367T (98.9 %) and Streptomyces phaeoluteigriseus DSM 41896T (98.8 %), but the strain formed a distinct clade in the phylogenetic tree. The DNA-DNA relatedness and average nucleotide identity values as well as evolutionary distances based on multilocus (atpD, gyrB, recA, rpoB and trpB) sequences between strain 13K301T and closely related type strains were significantly lower than the recommended threshold values. The cell wall contained ll-diaminopimelic acid and the whole-cell hydrolysates were glucose and ribose. Diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol were determined as the predominant polar lipids. The major menaquinones were identified as MK-9(H8) and MK-9(H6). On the basis of these genotypic and phenotypic data, it is proposed that strain 13K301T should be classified as representative of a novel species of the genus Streptomyces, for which the name Streptomyces cahuitamycinicus sp. nov. is proposed. The type strain is 13K301T (=DSM 106873T=KCTC 49110T). In addition, the whole genome-based comparisons as well as the multilocus sequence analysis revealed that the type strains of Streptomyces galilaeus and Streptomyces bobili belong to a single species. It is, therefore, proposed that S. galilaeus be recognised as a heterotypic synonym of S. bobili for which an emended description is given.

Genome-based classification of Micromonospora craterilacus sp. nov., a novel actinobacterium isolated from Nemrut Lake.

A novel actinobacterial strain, designated NA12T, was isolated from coastal sediment sample of Nemrut Lake, a crater lake in eastern Anatolia, Turkey. The taxonomic position of the strain was established using a polyphasic approach. Cultural and chemotaxonomic characteristics of the strain were consistent with its classification within the family Micromonosporaceae. The 16S rRNA gene sequence analysis of strain NA12T showed that the strain closely related to M. radicis AZ1-13T, M. zingiberis PLAI 1-1T, M. craniella LHW63014T and M. endophytica 202201T with pairwise sequence identity values ranging from 99.4 to 99.3%. Digital DNA-DNA hybridization values between strain NA12T and the closely related type strains were ranged from 41.0 to 18.3% while the average nucleotide identity values were between 87.3 and 86.5%, which are well below the designed cut-off points of 70 and 95%, respectively. The G + C content of genomic DNA was 71.5%. Whole-cell hydrolysates of strain NA12T contained 3-hydroxydiaminopimelic acid and meso-diaminopimelic acid. Cell-wall sugars were composed of arabinose, fucose, glucose, mannose, rhamnose and xylose. The polar lipid profile contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylglycerol, glycophospholipid, amino-phospholipid and two unidentified phospholipids. The predominant menaquinones were MK-9(H6) and MK-9(H4). Major fatty acids were iso-C16:0 and C17:1ω8c. Based upon the consensus of phenotypic and phylogenetic analyses as well as whole genome comparisons, strain NA12T (DSM 100982T = KCTC 39647T) is proposed to represent the type strain of a novel species, Micromonospora craterilacus sp. nov.

Micromonospora deserti sp. nov., isolated from the Karakum Desert.

An isolate, 13K206T, with typical morphological characteristics of the genus Micromonospora was obtained during a study searching for novel actinobacteria with biosynthetic potential from the Karakum Desert. A polyphasic approach was adopted to determine taxonomic affiliation of the strain. The strain showed chemotaxonomical properties consistent with its classification in the genus Micromonospora such as meso- and 3-OH-A2pm in the cell-wall peptidoglycan, xylose in whole-cell hydrolysate and diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol as major polar lipids. The results of phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain was closely related to 'Micromonospora spongicola' S3-1T, Micromonospora nigra DSM 43818T and Micromonospora yasonensis DS3186T with sequence similarities of 98.6, 98.5 and 98.4 %, respectively. Digital DNA-DNA hybridization and average nucleotide identity analyses in addition to gyrB gene analysis confirmed the assignment of the strain to a novel species within the genus Micromonospora for which the name Micromonospora deserti sp. nov. is proposed. The type strain is 13K206T (=JCM 32583T=DSM 107532T). The DNA G+C content of the type strain is 72.4 mol%.

Polyphasic classification of Nonomuraea strains isolated from the Karakum Desert and description of Nonomuraea deserti sp. nov., Nonomuraea diastatica sp. nov., Nonomuraea longispora sp. nov. and Nonomuraea mesophila sp. nov.

Five actinobacteria isolates, KC201T, KC401, KC310T, KC712T and 6K102T, were recovered from the Karakum Desert during an investigation of novel actinobacteria with biotechnological potential. A polyphasic approach confirmed the affiliation of the strains to the genus Nonomuraea. The strains showed chemotaxonomic and morphological properties consistent with their classification in the genus Nonomuraea. Furthermore, these strains clearly distinguished and formed well supperted clades in phylogenetic and phylogenomic trees. Low ANI and dDDH values and distinguishing phenotypic properties between isolates KC201T, KC310T, KC712T and 6K102T showed that these strains belonged to novel Nonomuraea species, the names proposed for these taxa are Nonomuraea deserti sp. nov., Nonomuraea diastatica sp. nov., Nonomuraea longispora sp. nov. and Nonomuraea mesophila sp. nov., with the type strains KC310T (=CGMCC 4.7331T =DSM 102919T =KCTC 39774T), KC712T (=CGMCC 4.7334T =DSM 102925T =KCTC 39776), KC201T (=CGMCC 4.7339T =DSM 102917T =KCTC 39781T) and 6K102T (=CGMCC 4.7541T =JCM 32916), respectively.

Micromonospora orduensis sp. nov., isolated from deep marine sediment.

A novel actinobacterial strain, designated S2509T, was isolated from marine sediment collected by a dredge at a depth of 45 m along Melet River offshore of the southern Black Sea coast, Ordu, Turkey. The cell wall peptidoglycan of strain was found to contain meso-diaminopimelic acid and 3-OH-diaminopimelic acid. The whole cell sugars detected were arabinose, glucose, rhamnose, ribose and xylose. The diagnostic phospholipids of strain S2509T were found to be diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, a glycolipid and two unidentified phospholipids. The predominant menaquinones were identified as MK-9(H8), MK-9(H6), MK-10(H8), MK-9(H4), MK-10(H4) and MK-10(H6). The major cellular fatty acids were found to be iso-C16:0, iso-C15:0 and 10-methyl C17:0. The taxonomic position of the strain was established using a polyphasic approach, showing that S2509T strain belongs to the genus Micromonospora. Phylogenetic analysis based on the 16S rRNA gene sequence of strain S2509T showed that it is closely related to the type strain of Micromonospora chokoriensis DSM 45160T (99.37% sequence similarity), and phylogenetically clustered with Micromonospora inaquosa LB39T (99.37%), Micromonospora lupini Lupac 14NT (99.16%), Micromonospora violae NEAU-zh8T (99.23%) and Micromonospora taraxaci NEAU-P5T (99.03%). The phylogenetic analysis based on the gyrB gene sequence of strain S2509T confirmed its close relationship with M. chokoriensis JCM 13247T (96.5% sequence similarity). Whole genome sequences confirmed by digital DNA-DNA hybridization analysis that the strain S2509T represents a novel species in the genus Micromonospora, for which the name Micromonospora orduensis sp. nov. is proposed. The type strain is S2509T (=DSM 45926T = KCTC 29201T).

Fast fluorometric enumeration of E. coli using passive chip.

In this report, a passive microfluidic chip design was developed for fast and sensitive fluorometric determination of Escherichia coli (E. coli) based on sandwich immunoassay. Initially, magnetic nanoparticles (MNPs) and chitosan modified mercaptopropionic acid capped cadmium telluride (CdTe) quantum dots (QDs) were functionalized with E.coli specific antibody to form a sandwich immunoassay with the E. coli. The magnetic separation and preconcentration of the E.coli from the sample solution was performed in the vial. Conjugation of QDs to the magnetically captured E. coli and washing were performed using a passive type of microchip. The microfluidic chip consists of four microchambers connected to each other by microchannels which act as capillary valves. Signal measurement was performed at the last chamber by using a hand-held spectrofluorometer equipped with a fiber optic reflection probe. The selectivity of the method was tested with Enterobacter aerogenes (E. aerogenes) and Salmonella enteritidis (S. enteritidis), it was observed that these bacteria have no interference effect on E.coli determination. The calibration curve was found to be linear in the range of 101-105 cfu/mL with a correlation coefficient higher than 0.99. The limit of detection was calculated as 5 cfu/mL. The method was successfully applied to spiked tap and lake water samples. The results suggest that the developed method is applicable for on-site E. coli detection and offers several advantages such as large dynamic range, high sensitivity, high selectivity and short analysis time.

The coupling of immunomagnetic enrichment of bacteria with paper-based platform.

In this study, the coupling of magnetic enrichment of bacteria from real samples with rapid surface enhanced Raman spectroscopy (SERS) detection was reported. The selective isolation and enrichment for the model bacteria Escherichia coli (E. coli) was performed using E. coli (primary) antibody bound-magnetic gold (Fe3O4@Au) nanoparticles. Following isolation and enrichment, the rennet enzyme was used to cleave of casein modified Fe3O4/Au-PEI nanoparticles from primary antibody-bound bacteria to prevent the nanoparticle aggregation and provide the movement of bacteria on nitrocellulose membrane. In the first part of the study, optimization studies were carried out namely; the amounts of gold nanoparticles (AuNPs), polyethyleneimine coated magnetic gold (Fe3O4/Au-PEI) nanoparticles, casein and rennet enzyme. The SERS signals of DTNB (5,5'-Dithiobis(2-nitrobenzoic acid)) molecule were collected on the test line and a calibration curve was plotted by using signal intensities. The correlation between the concentration of E. coli and SERS signal was found to be linear within the range of 101-107 cfu/mL (R2 = 0.984, LOD = 0.52 cfu/mL and LOQ = 1.57 cfu/mL). The selectivity of the paper-based lateral flow immunoassay (LFIA) was examined with Bacillus subtilis (B. subtilis), Micrococcus luteus (M. luteus), Salmonella enteritidis (S. enteritidis) which did not produce any significant response compared with E. coli measurement. Finally, the developed paper-based LFIA was tested with urine and milk samples. The obtained SERS results were compared with a plate counting method results which were in a good accordance. The developed method was found as rapid and sensitive to E. coli with a total analysis time of less than 60 min.

SERS-based rapid assay for sensitive detection of Group A Streptococcus by evaluation of the swab sampling technique.

Beta-hemolytic, Group A Streptococcus pyogenes (GAS) is a life-threating pathogen and the reason for prominent disease, pharyngitis. The conventional analysis of GAS, gold standard, takes 48 hours and the related rapid tests lack in accuracy and sensitivity. In this study, firstly, the efficiency of swab sampling, which is a must in the GAS detection, was discussed with the proposed surface-enhanced Raman spectroscopy (SERS)-based batch assay and each step was controlled by the plate-counting method. Secondly, SERS-based lateral flow immunoassay (LFIA) test strips were constructed and the variation in the SERS intensity of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) was observed. Thus, a linear correlation was found with a R2 value of 0.9926 and the LOD was calculated to be 0.2 CFU mL-1 of GAS which could be counted as one cell. The combination of the gold standard with the LFIA-SERS technique enabled the fast and accurate pathogen detection. In addition, GAS was quantified with paper-based test strips up to 100 CFU ml-1 level of bacteria for the first time without any interference. Besides, this study was featured with the discussion of the whole cell and pretreated cell detection of pathogens with LFIAs. Therefore, this work enlightens the points that have never been discussed on pathogen detection with paper-based platforms.