In vitro metabolism of the emerging contaminant 6PPD-quinone in human and rat liver microsomes: Kinetics, pathways, and mechanism.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q) is an ozonation product of the rubber antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD). 6PPD-Q has recently been detected in various environmental media, which may enter the human body via inhalation and skin contact pathways. However, the human metabolism of 6PPD-Q has remained unknown. This study investigated the in vitro Cytochrome P450-mediated metabolism of 6PPD-Q in human and rat liver microsomes (HLMs and RLMs). 6PPD-Q was significantly metabolized at lower concentrations but slowed at high concentrations. The intrinsic clearance (CLint) of 6PPD-Q was 21.10 and 18.58 µL min-1 mg-1 protein of HLMs and RLMs, respectively, suggesting low metabolic ability compared with other reported pollutants. Seven metabolites and one intermediate were identified, and metabolites were predicted immunotoxic or mutagenic toxicity. Mono- and di-oxygenation reactions were the main phase I in vitro metabolic pathways. Enzyme inhibition experiments and molecular docking techniques were further used to reveal the metabolic mechanism. CYP1A2, 3A4, and 2C19, especially CYP1A2, play critical roles in 6PPD-Q metabolism in HLMs, whereas 6PPD-Q is extensively metabolized in RLMs. Our study is the first to demonstrate the in vitro metabolic profile of 6PPD-Q in HLMs and RLMs. The results will significantly contribute to future human health management targeting the emerging pollutant 6PPD-Q.

Immune response and protective efficacy of Streptococcus agalactiae vaccine coated with chitosan oligosaccharide for different immunization strategy in nile tilapia (Oreochromis niloticus).

In the past decade, the outbreak of Streptococcus agalactiae has caused significant economic losses in tilapia farming. Vaccine immunization methods and strategies have gradually evolved from single-mode to multi-mode overall prevention and control strategies. In this study, an inactivated vaccine of S. agalactiae with a chitosan oligosaccharide (COS) adjuvant was constructed using different administration methods: intraperitoneal injection (Ip), immersion combined with intraperitoneal injection (Im + Ip), immersion combined with oral administration (Im + Or), and oral administration (Or). Safety analysis revealed no adverse effects on tilapia, and the vaccine significantly promoted fish growth and development when administered through Im + Or or Or immunization. Following vaccination, innate immunity parameters including SOD, ACP and CAT activities were all significantly enhanced. Additionally, specific serum IgM antibodies reached their highest level at the 6th week post vaccination. Skin and intestinal mucus IgT antibodies reached peaked at the 6th and 7th week post vaccination, respectively. The relative peak expression values for IL-8, IL-12, MHC-I, MHC-II, IgM, IgT, CD4, CD8, TNFα, IFNγ from Im + Ip group were significantly higher than those in Ip group, Im + Or group and Or group in most cases (p < 0.05). Importantly, the relative protection survival of Im + Ip group was the highest (78.6%), followed by the Ip group (71.4%), the Or group (64.3%) and the Im + Or group (57.1%). In summary, this study encourages further research on multi-channel immunization strategies of other kinds of vaccines in other aquatic economic animals to improve their disease resistance.

Synergistic Reinforcement of Si3N4 Based Ceramics Fabricated via Multiphase Strengthening under Low Temperature and Short Holding Time.

Si3N4 ceramic as a tool material shows promising application prospects in high-speed machining fields; however, the required high mechanical properties and low-cost preparation of Si3N4 ceramic tool materials restrict its application. Herein, synergistic reinforced Si3N4 ceramic tool materials were fabricated by adding ß-Si3N4 seeds, inexpensive Si3N4 whiskers and TiC particles into coarse commercial Si3N4 powder (D50 = 1.5 µm), then sintering by hot-pressing with low temperature and short holding time (1600 °C-30 min-40 MPa). The phase assemblage, microstructure evolution and toughening mechanisms were investigated. The results reveal that the sintered Si3N4 ceramics with synergistic reinforcement, compared to those with individual reinforcement, present an enhancement in relative density (from 94.92% to 97.15%), flexural strength (from 467.56 ± 36.48 to 809.10 ± 45.59 MPa), and fracture toughness (from 8.38 ± 0.19 to 10.67 ± 0.16 MPa·m1/2), as well as a fine Vickers hardness of 16.86 ± 0.19 GPa. Additionally, the various reinforcement modes of Si3N4 ceramics including intergranular fracture, crack deflection, crack bridging and whiskers extraction were observed in crack propagation, arising from the contributions of the added ß-Si3N4 seeds, Si3N4 whiskers and TiC particles. This work is expected to serve as a reference for the production of ceramic cutting tools.

Detrimental Association Between Blood Cadmium Concentration and Trabecular Bone Score.

Osteoporosis has been recognized as a significant cause of disability in the elderly leading to heavy socioeconomic burden. Current measurements such as dual-energy X-ray absorptiometry (DEXA) and bone mineral density (BMD) have limitations. In contrast, trabecular bone score (TBS) is an emerging tool for bone quality assessment. The objective of our study was to investigate the relationship between TBS and trace elements (cadmium and lead). We analyzed all subjects from the 2005-2006 and 2007-2008 National Health and Nutrition Examination Survey (NHANES) dataset and included a total of 8,244 participants in our study; 49.4% of the enrolled subjects were male. We used blood cadmium (Cd) and lead (Pb) concentrations to define environmental exposure. The main variables were TBS and BMD. Other significant demographic features were included as covariates and later adjusted using linear regression models to determine the association between TBS and four quartiles based on the blood trace element concentrations with or without sex differences. The fully adjusted regression model revealed a negative relationship between TBS and blood cadmium (B-Cd) significant for both males and females (both p < 0.05). The ß-coefficient for males was -0.009 (95% confidence intervals (CI): (-0.015 to -0.004)) and -0.019 for female (95% CI: (-0.024 to -0.013)). We also found a dose-dependent relationship between TBS and B-Cd for both sexes (both trend's p < 0.05). Our study concluded that TBS could measure Cd-related bone quality deterioration for both males and females.

Clinical Relevance of Serum Klotho Concentration and Sagittal Abdominal Diameter.

Klotho is an anti-aging gene. Studies have revealed its association with insulin resistance. Visceral fat is related to insulin resistance, and the sagittal abdominal diameter (SAD) can serve as a biomarker for visceral fat (VF). This study investigated the association between SAD and serum Klotho concentration (SKC). We enrolled 2301 participants from the 2011−2012 National Health and Nutrition Examination Survey (NHANES) dataset, and 49.2% of the enrolled individuals were male. Qualified participants were separated into four quartiles according to the SAD value. SKC values were obtained by ELISA. Demographic characteristics, body mass index (BMI), systolic blood pressure, and biochemistry parameters with significance were analyzed using multivariate linear regression models. The mean age of the study participants was 57.22 ± 10.53 years. The fully adjusted regression model showed a negative association between SAD and SKC (p < 0.05), with a ß-coefficient of −12.02. We also analyzed subgroups of participants according to age and BMI. Participants with an age ≥65 and <65 years old were each negatively associated with SKC, and this association was significant for participants with a BMI ≥ 30 kg/m2 (p = 0.001, ß-coefficient: −18.83). We also found a concentration-dependent relationship between SAD and SKC. In conclusion, VF and SKC are associated, and SAD can serve as a surrogate of VF and an indicator of SKC.

Effects of Micro-Arc Oxidation Discharge Parameters on Formation and Biomedical Properties of Hydroxyapatite-Containing Flower-like Structure Coatings.

The micro-arc oxidation (MAO) process was used to prepare hydroxyapatite-containing flower-like structure coatings on commercially pure titanium substrates with various values of the applied voltage (330, 390, 450 V), applied current (0.4, 0.5, 0.6 A), and duration time (1, 3, 5 min). It was found that the surface morphology of the coatings was determined primarily by the applied voltage. A voltage of 330 V yielded a flower-like/plate-like structure, while voltages of 390 V and 450 V produced a flower-like structure and a porous morphology, respectively. The applied current and duration time mainly affected the coating formation speed and petal size of the flower-like structures, respectively. The coatings prepared using voltages of 330 V and 390 V (0.6 A, 5 min) both contained Ti, TiO2-A (anatase), TiO2-R (rutile), DCPD (CaHPO4·2H2O, calcium hydrogen phosphate), and hydroxyapatite (HA). However, the latter coating contained less DCPD and had a higher HA/DCPD ratio and a Ca/P ratio closer to the ideal value of HA. The coating prepared with a voltage of 450 V consisted mainly of Ti, TiO2-A, TiO2-R, and CaTiO3. For the coatings prepared with a voltage of 390 V, the flower-like structures consisted mainly of HA-containing compounds. DCPD plate-like structures were observed either between the HA-containing flower-like structures (330 V samples) or within the flower-like structures themselves (390 V samples). The coating surfaces with flower-like/plate-like or flower-like structures had a greater roughness, which increased their hydrophilicity and resulted in superior bioactivity (SBF immersion) and biocompatibility (MG-63 cell culture). The optimal biomedical performance was found in the 390 V coating due to its flower-like structure and high HA/DCPD ratio.

Uptake, elimination, and toxicokinetics of selected pharmaceuticals in multiple tissues of Nile tilapia (Oreochromis niloticus) exposed to environmentally relevant concentrations.

Pharmaceuticals in aquatic environment displayed adverse effects to fish. The effects are usually related to the internal levels of pharmaceuticals accumulated in specific fish tissues. In this study, we investigated the uptake, elimination, and toxicokinetics of six pharmaceuticals, e.g. naproxen (NAX), diclofenac (DCF), ibuprofen (IBU), carbamazepine (CBZ), fluoxetine (FLX), and sertraline (SER), in 11 fish tissues of Nile tilapia. The experiments were conducted in a flow-through system with an 8-day uptake/8-day elimination periods. The fish exposure groups involved the control, single FLX, and mixture of six pharmaceuticals at environmentally relevant concentration of 4 µg/L. FLX and SER showed the maximum concentrations of 145 and 201 ng/g wet weight, respectively, in fish spleen tissue, while NAX and IBU were not detected in any tissue. The mean concentrations for the pharmaceuticals in Nile tilapia tissues generally followed the order: bile> kidney, gut, stomach, liver> brain, gill, spleen> plasma, skin, muscle. The steady-state bioconcentration factors in various tissues generally range at 0.74-437.58 L/kg. The uptake and elimination toxicokinetics illustrated the rapid accumulation and depuration of pharmaceuticals in fish tissues. The results help to understand the internal bioconcentration, tissue distribution, and toxicokinetics of pharmaceuticals in multiple fish biological compartments.

Case Report: In Situ Vaccination by Autologous CD16+ Dendritic Cells and Anti-PD-L 1 Antibody Synergized With Radiotherapy To Boost T Cells-Mediated Antitumor Efficacy In A Psoriatic Patient With Cutaneous Squamous Cell Carcinoma.

The combination of radiotherapy and immunotherapy improves the survival rate of patients with malignancies developed through escape from T-cell-mediated immune surveillance. Immune checkpoint inhibitors, such as anti-programmed cell death protein-ligand 1 (anti-PD-L1) antibody, are used to rescue exhausted T cells. Simultaneously, dendritic cells (DCs) which are antigen-presenting cells that can initiate T-cell activation, are used to induce a tumor-specific immune response. However, the synergistic antitumor efficacy of the aforementioned combinational immunotherapy with intratumoral injection of low-dose DCs has not been reported, and the underlying therapeutic mechanism requires further investigation. Herein, we present the special case of a psoriatic patient with cutaneous squamous cell carcinoma (cSCC) in the right inguinal region, these two diseases characterized by opposing contradiction, further complicating treatments and side-effect management efforts. To treat the intractable SCC without exaggerating psoriasis, we developed the triple-regimen therapy (TRT) with the intratumoral injection of low-dose autologous DCs and anti-PD-L1 combined with radiotherapy. The injected DCs were obtained simply through leukapheresis without prior G-CSF administration for mobilization nor tumor-antigen loading for expansion. The patient received three radiation doses (24, 18, and 18 Gy) combined with three intratumoral injections of anti-PD-L1 antibody (40, 60, and 120 mg) plus autologous DCs (80% of the DC subpopulation being CD16+ myeloid DC with approximate amounts of 7.3 × 104, 2.5 × 106, and 1.7 × 107) within 10 weeks. The efficacy of the TRT was encouraging in shrinking tumor mass with remarkable SUVmax reduction (approximately 42%) on FDG PET-Scan despite relatively low-dose DCs were available. The low-dose intratumoral immunotherapy induced mild cutaneous side effects as expected. The transcriptomes were compared between pre-TRT and post-TRT biopsies to analyze underlying mechanical pathways of the TRT protocol. Over 10 highly significantly enriched T-cell-related pathways (P <0.0001) were identified in post-TRT biopsies. In addition, the activation of both innate and adaptive immunity was significantly enriched in post-TRT peripheral blood samples. We develop the easily accessible TRT which produces both local anti-tumor T-cell responses and systemic antitumor immunity for treating cSCC patients, especially for those with autoimmune disease.

Spraying rhubarb powder solution under gastroscope in the treatment of acute non-varicose upper gastrointestinal bleeding: A systematic review and meta-analysis of randomized controlled trials.

OBJECTIVE: Systematically assessing the safety and effectiveness of spraying rhubarb powder solution under gastroscope for the treatment of acute non-varicose upper gastrointestinal bleeding, and confirmation for further clinical research and application. METHODS: We searched the following databases up till November 2019: PubMed, the Cochrane Library, EMBASE, CNKI, WanFang Data, VIP and SinoMed. Randomized controlled trials (RCTs) were used to compare the curative effect of spraying rhubarb powder solution with other drugs under gastroscope for the treatment of acute non-varicose upper gastrointestinal bleeding. RESULTS: Out of 171 articles, 14 RCTs involving 1493 patients were included. All control groups included in the RCTs were treated with norepinephrine solution. The hemostatic effect of spraying rhubarb powder solution under gastroscope was examined for 24 h at high concentration (0.1 g/mL). The hemostatic effect at higher conc. (0.1 g/mL) found far more better than low conc.(RR = 1.48;95 %CI:1.25,1.75;P﹤0.00001) (0.03 g/mL)as homeostatic effect at low conc.is same that of norepinephrine solution (RR = 1.02;95 %CI:0.94,1.10;P = 0.62). Moreover within 48 h, rhubarb powder solution with 0.1 g/mL or 0.15 g/mL conc. have of significantly higher hemostatic effects than norepinephrine solution (RR = 1.18;95 % CI: 1.08, 1.30;P = 0.0003). Occurrence of rebleeding event within 48 h after successful hemostasis (RR = 0.42;95 %CI:0.24,0.74;P = 0.003) reduced exceptionally. After that the hemostatic effect of rhubarb powder solution with 0.1 g/mL conc.examined within 72 h again exhibited significant improvement than norepinephrine solution (RR = 1.19;95 %CI:1.12,1.26;P < 0.00001). On par with immediate hemostasis time, rhubarb powder solution took unprecedented less time than norepinephrine solution;(MD=-5.56S;95 %CI:-6.16, -4.95;P﹤0.00001). Additionally, the adverse reaction produced by rhubarb powder solution is much lower than norepinephrine solution (RR = 0.22;95 %CI:0.11,0.42;P < 0.00001). CONCLUSIONS: According to meta-analysis, Spraying rhubarb powder solution under gastroscope in the treatment of acute non-varicose upper gastrointestinal bleeding is superior to norepinephrine solution in improving hemostasis effect. Shortening immediate hemostasis time and reducing rebleeding,and is safe to use. Based on the results of this study, physicians can treat patients with acute non-varicose upper gastrointestinal bleeding by spraying rhubarb powder solution under gastroscope according to the patients' condition.However, the sample size included in this study is small and of substandard quality qu, and a large sample size clinical trial with strict design and normative report is needed to verify the safety and efficacy of rhubarb powder solution under gastroscope for acute non-varicose upper gastrointestinal bleeding.

Locating secret messages based on quantitative steganalysis.

Steganography poses a serious challenge to forensics because investigators cannot identify even traces of secret messages embedded using a steganographer. Contrarily, the objective of locating steganalysis is to locate the embedded message, which should help extract the secret message. In this paper, a methodology of locating steganalysis using quantitative steganalysis is presented for multiple stego images with embedded messages along the same embedding path. Three typical quantitative steganalysis methods are applied to the methodology to locate the messages embedded using LSB re-placement. Experimental results show that the presented methods can reliably estimate the embedding positions, which verifies the validity of the presented methodology. The presented methodology points out a new use of quantitative steganalysis, and further demonstrates that it is necessary to design more precise quantitative steganalysis methods.

Pedobacter helvus sp. nov., isolated from farmland soil.

A strictly aerobic, Gram-stain-negative, rod-shaped, yellow, non-spore-forming bacterial strain, designated P-25T, was isolated from soil collected in Yantai, Shandong Province, PR China. The temperature, pH and NaCl concentration ranges for the growth of strain P-25T were 10-37 °C (optimum, 28-30 °C), pH 6.0-9.0 (optimum, pH 7.5-8.0) and 0-4 % (w/v) (optimum, 1 % w/v), respectively. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain P-25T was most closely related to Pedobacter xixiisoli S27T (98.1 % 16S rRNA gene sequence similarity), followed by Pedobacter chitinilyticus CM134L-2T (97.2 %) and Pedobacter ureilyticus THG-T11T (97.1 %). The genomic DNA G+C content of strain P-25T based on its draft genome sequence was 38.1 %. MK-7 was the major respiratory quinone, and iso-C15 : 0, C16 : 1ω7c and/or C16 : 1ω6c (summed feature 3) and iso-C17 : 0 3-OH were the major fatty acids. The major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified phospholipid, two unidentified lipids, five unidentified aminolipids and two unidentified glycolipids. Average nucleotide identity values for the draft genomes between strain P-25T and strains S27T, CM134L-2T and THG-T11T were 81.8, 77.6 and 81.2 %, respectively, and the digital DNA-DNA hybridization (dDDH) values were 30.0, 19.2 and 27.6 %, respectively. Based on their phylogenetic and phenotypic characteristics, chemotaxonomic data, and dDDH results, strain P-25T is considered to represent a novel species of the genus Pedobacter, for which the name Pedobacter helvus sp. nov. is proposed; the type strain is strain P-25T (KCTC 62821T=CCTCC AB 2018185T).

Steroidobacter soli sp. nov., isolated from farmland soil.

A Gram-stain-negative bacterial strain, designated JW-3T, was isolated from a soil sample collected from farmland in Yantai, Shandong Province, PR China. Cells of strain JW-3T are motile rods and strictly aerobic, showing catalase- and oxidase-positive reactions. Strain JW-3T could grow at 16-37 °C (optimum, 30 °C), at pH 6.0-9.0 (pH 7.0) and in the presence of 0-1 % (w/v) NaCl (0.5 %, in Luria-Bertani broth). The major fatty acids were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c; 35.5 %), iso-C16 : 0 (16.7 %) and C12 : 0 (10.8 %). The major respiratory quinone was ubiquinone-8 (Q8). The polar lipids of strain JW-3T consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, four unidentified phospholipids, two unidentified lipids, two unidentified glycolipids and a partial unidentified aminophospholipid. Strain JW-3T was most closely related to Steroidobacter agariperforans KA5-BT with 97.67 % 16S rRNA gene sequence similarity. Results of phylogenetic analyses, based on 16S rRNA gene sequencing, showed that strain JW-3T forms a distinct phylogenic lineage within the genus Steroidobacter of the family Sinobacteraceae. The DNA G+C content of strain JW-3T was 62.57 mol%, based on its draft genome sequence. Average nucleotide identity values and digital DNA-DNA hybridization values for draft genomes, between strain JW-3T and strain KA5-BT, were 84.54 and 30.80 %, respectively. Based on its phenotypic, chemotaxonomic and molecular features, and DNA-DNA hybridization results, strain JW-3T represents a novel species of the genus Steroidobacter, for which the name Steroidobactersoli sp. nov. is proposed. The type strain is JW-3T (=CCTCC AB 2018184T=KCTC 62820T).

Chitinophaga deserti sp. nov., isolated from desert soil.

An aerobic bacterial strain, designated XJ-2T, was isolated from a soil sample collected from Gurbantunggut Sandy Desert in PR China. Cells of strain XJ-2T were Gram-stain-negative, non-motile, rod-shaped and non-spore-forming. The new isolate grew well at 10-37 °C (optimum, 28-30 °C), pH 6.0-11.0 (pH 7.0) and 0-1 % (w/v) NaCl (0 %). The 16S rRNA gene sequence of strain XJ-2T showed the highest similarity to that of Chitinophaga rhizosphaerae T16R-86T (99.0 %), followed by Chitinophaga barathri YLT18T (97.0 %), Chitinophagahumicola Ktm-2T (96.7 %) and Chitinophaga niabensis JS13-10T (96.4 %). The major menaquinone of strain XJ-2T was menaquinone 7 and the predominant fatty acids (>5 %) were iso-C15 : 0, C16 : 1ω5c and iso-C17 : 0 3-OH. The polar lipids consisted of phosphatidylethanolamine, an unidentified glycolipid, three unidentified aminolipids and five unidentified lipids. The genome size was 6.33 Mb, comprising 5268 predicted genes with a G+C content of 41.5 mol%. The DNA G+C content was 50.5 mol% based on total genome calculations. The average nucleotide identity and the digital DNA-DNA hybridization values between strain XJ-2T and strain T16R-86T were 79.6 and 22.3 %, respectively. DNA-DNA relatedness between strain XJ-2T and strain YLT18T was 17.0 %. Based on the physiological, biochemical and chemotaxonomic characteristics, strain XJ-2T represents a novel species of the genus Chitinophaga, for which the name Chitinophagadeserti sp. nov. is proposed. The type strain is XJ-2T (KCTC 62443T=CCTCC AB 2018019T).

The functional verification of EGFR-CAR T-cells targeted to hypopharyngeal squamous cell carcinoma.

BACKGROUND: The aim of this study was to validate the antitumor function of EGFR-chimeric antigen T-cells (CART) targeted to FaDu cells, a hypopharyngeal squamous cell carcinoma cell line, and to provide a preclinical basis for the application of CART cell technology in hypopharyngeal squamous cell carcinoma. METHODS: Detection of cytokine secretions of EGFR-CAR T and CART-controls in the presence of target cells and nontarget cells as an indicator of CART cell activation. Detection of the cytotoxic effects of EGFR-CAR T on specific tumors in the presence of target cells was evaluated by LDH release and CART cell proliferation. RESULTS: The results showed that cytokine secretion increased significantly after EGFR-CAR T-cells were incubated with target cells, and EGFR-CAR T-cells has higher cytotoxic effect on target cells than the CART-control group. The target cell lysis rate was 52.66%. The proliferation of EGFR-CAR T-cells in the presence of target cells was not distinctly observed. CONCLUSION: In this study, we validated the antitumor function of EGFR-CAR T-cells targeted to the FaDu cell line and provided the foundation for application of the CART technique in the treatment of hypopharyngeal carcinoma.

Aquamicrobium soli sp. nov., a bacterium isolated from a chlorobenzoate-contaminated soil.

An aerobic, Gram-stain negative, short rod-shaped, asporogenous, non-motile bacterium designated strain NK8T was isolated from a chlorobenzoate contaminated soil in China. Strain NK8T was observed to grow optimally at pH 7.0, 30 °C and in the absence of NaCl in LB medium. The G + C content of the total DNA of strain NK8T was found to be 65.5 mol%. The 16S rRNA gene sequence of strain NK8T showed high similarity to that of Aquamicrobium aerolatum Sa14T (97.3%), followed by Aquamicrobium lusatiense S1T (96.7%) and Mesorhizobium sangali SCAU7T (96.6%). The DNA-DNA relatedness between strain NK8T and A. aerolatum Sa14T was 35.5 ± 0.9%. The major fatty acids of strain NK8T were determined to be C19:0 cyclo ω8c (45.6%), C18:1 ω7c (33.4%) and C16:0 (8.4%). The respiratory quinone was found to be ubiquinone Q-10. The major polyamine was found to be spermidine. The polar lipid profile include the major compounds phosphatidylcholine and diphosphatidylglycerol, and moderate amounts of phosphatidylethanolamine, phosphatidylmonomethylethanolamine, aminolipid and phospholipid. Based on the differential biochemical and physiological characteristics, the geno-, chemo- and phenotypic characteristics, strain NK8T is proposed to represent a novel species of the genus Aquamicrobium, Aquamicrobium soli sp. nov. The type strain is NK8T (=KCTC 52165T=CCTCC AB2016045T).

Characterisation of fosfomycin resistance mechanisms and molecular epidemiology in extended-spectrum ß-lactamase-producing Klebsiella pneumoniae isolates.

Although fosfomycin is a treatment option for infections caused by extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae, fosfomycin resistance has been documented. To our knowledge, fosfomycin resistance mechanisms in Klebsiella pneumoniae have not been systematically investigated. A total of 108 ESBL-producing K. pneumoniae isolates collected from Kaohsiung Medical University Hospital, Taiwan, from August 2012 to May 2013 were analysed in this study. Pulsed-field gel electrophoresis (PFGE) revealed 64 pulsotypes and six non-typeable isolates, indicating high genetic diversity. Moreover, pulsotypes V (n = 6), VII (n = 11) and LI (n = 4) belonging to ST11 were major types. Among 30 (27.8%) fosfomycin-non-susceptible isolates, 21 (70%) had a MurA amino acid substitution, and seven new variations increased the fosfomycin minimum inhibitory concentration (MIC) by 8- to 16-fold compared with wild-type MurA in Escherichia coli DH5α.strain. Functionless transporters (GlpT and UhpT) with various mutations were found in 29 isolates (97%). No known fosfomycin-modifying enzymes were detected in this study. The major resistance mechanisms to fosfomycin in K. pneumoniae were amino acid variations in the drug target and transporters.

Characterization of Fosfomycin Resistant Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolates from Human and Pig in Taiwan.

To investigate the efficacy of fosfomycin against extended-spectrum ß-lactamases (ESBL) producing Escherichia coli in Taiwan and the resistance mechanisms and characterization of human and pig isolates, we analyzed 145 ESBL-producing isolates collected from two hospitals (n = 123) and five farms (n = 22) in Taiwan from February to May, 2013. Antimicrobial susceptibilities were determined. Clonal relatedness was determined by PFGE and multi-locus sequence typing. ESBLs, ampC, and fosfomycin resistant genes were detected by PCR, and their flanking regions were determined by PCR mapping and sequencing. The fosfomycin resistant mechanisms, including modification of the antibiotic target (MurA), functionless transporters (GlpT and UhpT) and their regulating genes such as uhpA, cyaA, and ptsI, and antibiotic inactivation by enzymes (FosA and FosC), were examined. The size and replicon type of plasmids carrying fosfomycin resistant genes were analyzed. Our results revealed the susceptibility rates of fosfomycin were 94% for human ESBL-producing E. coli isolates and 77% for pig isolates. The PFGE analysis revealed 79 pulsotypes. No pulsotype was found existing in both human and pig isolates. Three pulsotypes were distributed among isolates from two hospitals. ISEcp1 carrying blaCTX-M-group 9 was the predominant transposable elements of the ESBL genes. Among the thirteen fosfomycin resistant isolates, functionless transporters were identified in 9 isolates. Three isolates contained novel amino acid substitutions (Asn67Ile, Phe151Ser and Trp164Ser, Val146Ala and His159Tyr, respectively) in MurA (the target of fosfomycin). Four isolates had fosfomycin modified enzyme (fosA3) in their plasmids. The fosA3 gene was harboured in an IncN-type plasmid (101 kbp) in the three pig isolates and an IncB/O-type plasmid (113 kbp) in the human isolate. In conclusion, we identified that 6% and 23% of the ESBL-producing E. coli from human and pigs were resistant to fosfomycin, respectively, in Taiwan. No clonal spread was found between human and pig isolates. Functionless transporters were the major cause of fosfomycin resistance, and the fosA3-transferring plasmid between isolates warrants further monitoring.

Camelimonas fluminis sp. nov., a cyhalothrin-degrading bacterium isolated from river water.

An aerobic, Gram-stain-negative, short rod-shaped, non-spore-forming, cyhalothrin-degrading bacterial strain, XZ2(T), was isolated from the surface water of Hanjiang River in Wuhan, China. Strain XZ2(T) grew optimally at pH 6.0, 30 °C and in the absence of NaCl. The G+C content of the total DNA was 64.1 mol%. The 16S rRNA gene sequence of strain XZ2(T) showed the highest similarity to that of Camelimonas lactis M 2040(T) (99.1%), followed by Camelimonas abortus UK34/07-5(T) (95.9%) and Chelatococcus daeguensis K106(T) (95.3%). The major cellular fatty acids of strain XZ2(T) were C19 : 0 cyclo ω8c (63.1%), C16 : 0 (15.0%) and C18 : 1ω7c/C18 : 1ω6c (summed feature 8; 8.9%). C18 : 0 3-OH was also detected as the major hydroxylated fatty acid. The respiratory quinone was ubiquinone Q-10. The polar lipid profile included the major compounds phosphatidylcholine and diphosphatidylglycerol, and moderate amounts of phosphatidylethanolamine, phosphatidylglycerol and two unidentified aminolipids. The predominant compound in the polyamine pattern was spermidine. These chemotaxonomic data supported the affiliation of strain XZ2(T) to the genus Camelimonas. The DNA-DNA hybridization value between strain XZ2(T) and Camelimonas lactis M 2040(T) was 43.5 ± 0.6%. DNA-DNA hybridization data as well as biochemical and physiological characteristics strongly supported the genotypic and phenotypic differentiations between strain XZ2(T) and Camelimonas lactis M 2040(T). Therefore, strain XZ2(T) represents a novel species of the genus Camelimonas, for which the name Camelimonas fluminis sp. nov. is proposed. The type strain is XZ2(T) ( = KCTC 42282(T) = ACCC 19738(T)).

The contribution of antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates: an emphasis on efflux pump activity.

Due to the limited information of the contribution of various antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates, Antibiotic resistance mechanisms, including integron analysis, identification of quinolone resistance-determining region mutations, measurement of efflux pump activity, and sequence analysis of efflux pump regulators, were investigated in 66 clinical B. cepacia complex isolates. Species were identified via recA-RFLP and MALDI-TOF. Four genomovars were identified by recA-RFLP. B. cenocepacia (genomovar III) was the most prevalent genomovar (90.1%). Most isolates (60/66, 90.9%) were correctly identified by MALDI-TOF analysis. Clonal relatedness determined by PFGE analysis revealed 30 pulsotypes, including two major pulsotypes that comprised 22.7% and 18.2% of the isolates, respectively. Seventeen (25.8%) isolates harboured class 1 integron with various combinations of resistance genes. Among six levofloxacin-resistant isolates, five had single-base substitutions in the gyrA gene and three demonstrated efflux pump activities. Among the 42 isolates exhibiting resistance to at least one antimicrobial agent, 94.4% ceftazidime-resistant isolates (17/18) and 72.7% chloramphenicol-resistant isolates (16/22) demonstrated efflux pump activity. Quantitation of efflux pump RNA level and sequence analysis revealed that over-expression of the RND-3 efflux pump was attributable to specific mutations in the RND-3 efflux pump regulator gene. In conclusion, high-level expression of efflux pumps is prevalent in B. cepacia complex isolates. Mutations in the RND-3 efflux pump regulator gene are the major cause of efflux pump activity, resulting in the resistance to antibiotics in clinical B. cepacia complex isolates.