Development and evaluation of a rapid and sensitive multienzyme isothermal rapid amplification with a lateral flow dipstick assay for detection of Acinetobacter baumannii in spiked blood specimens.

Purpose: This study aimed to establish the multienzyme isothermal rapid amplification with a lateral flow dipstick (MIRA-LFD) assay and evaluate its performance in detection of A. baumannii in spiked blood specimens. Methods: The study was divided into two stages: a pilot study to establish the methodology and a clinical validation study to evaluate its performance. In the first step, we designed primers specific to detect A. baumannii, optimized the MIRA-LFD assay and analyzed its performance regarding limits of detection, reproducibility, specificity, and efficiency of detection using real-time PCR method. In the second step, we obtained 50 spiked blood isolates and detected these pathogens by MIRA-LFD assay. The MIRA-LFD time was 15 min from DNA sample amplification to complete pathogen detection. Results: The developed MIRA-LFD assay displayed a detection limit of 6 CFU/mL for detecting A. baumannii, which was significantly better than that of real-time PCR method, and no cross-reactivity was observed in other non-A. baumannii studied. The results obtained with 50 spiked blood isolates suggested that the developed MIRA-LFD assay had high specificity and sensitivity for identifying A. baumannii. Conclusions: This study demonstrates that the established MIRA-LFD assay is time-saving, more effective and sensitive, which may become a powerful tool for rapid and reliable diagnosis of bloodstream infection caused by A. baumannii in primary hospitals.

Understanding the Bacterial Response to Mycotoxins: The Transcriptomic Analysis of Deoxynivalenol-Induced Changes in Devosia mutans 17-2-E-8.

Deoxynivalenol (DON) is a major fusarium toxin widely detected in cereal grains. The inadvertent exposure to this fungal secondary-metabolite gives rise to a myriad of adverse health effects including appetite loss, emesis, and suppression of the immune system. While most of the attention this mycotoxin has gained in the past four decades was related to its eukaryotic toxicity (monogastric animals and plants more precisely), recent studies have begun to reveal its negative influence on prokaryotes. Recently presented evidence indicates that DON can negatively affect many bacterial species, raising the possibility of DON-induced imbalances within the microbiota of the human and animal gut, in addition to other environmental niches. This in turn has led to a greater interest in understanding bacterial responses toward DON, and the involved mechanism(s) and metabolic pathways, in order to build a more comprehensive picture of DON-induced changes in both prokaryotes and eukaryotes alike. This study reveals the transcriptomic profiling of Devosia mutans strain 17-2-E-8 after the inclusion of DON within its growth medium. The results highlight three adaptive mechanisms involved in the response of D. mutans 17-2-E-8 to this mycotoxin, which include: (a) activation of adenosine 5'-triphosphate-binding cassette transporters; (b) engagement of a toxin-specific pyrroloquinoline quinone-dependent detoxification pathway; and finally (c) the upregulation of auxiliary coping proteins such as porins, glutathione S-transferases, and phosphotransferases. Some of the identified mechanisms are universal in nature and are shared with other bacterial genera and species.

The enzymatic epimerization of deoxynivalenol by Devosia mutans proceeds through the formation of 3-keto-DON intermediate.

The enzymatic detoxification of deoxynivalenol (DON) is a promising mitigation strategy for addressing this mycotoxin contamination of cereal grains. A recently described bacterium, Devosia mutans 17-2-E-8, capable of transforming DON into its non-toxic stereoisomer 3-epi-DON, holds promise for the development of such applications. Earlier observations suggested that DON epimerization proceeds via a two-step catalysis with 3-keto-DON as an intermediate. The results of this study indicate that NADPH is required for DON epimerization by cell-free protein extracts of D. mutans, while high concentrations of glucose and sucrose have a suppressive effect. Chemically synthesized 3-keto-DON incubated with D. mutans protein fractions enriched by ammonium sulfate precipitation at 35-55% saturation selectively reduced 3-keto-DON to 3-epi-DON, but fell short of supporting the complete epimerization of DON. In addition, seven Devosia species investigated for DON epimerization were all able to reduce 3-keto-DON to 3-epi-DON, but only a few were capable of epimerizing DON. The above observations collectively confirm that the enzymes responsible for the oxidation of DON to 3-keto-DON are physically separate from those involved in 3-keto-DON reduction to 3-epi-DON. The enzymatic nature of DON epimerization suggests that the process could be used to develop genetically engineered crops or microorganisms, ultimately reducing foodborne exposure of consumers and farm animals to DON.

Microbial biotransformation of DON: molecular basis for reduced toxicity.

Bacteria are able to de-epoxidize or epimerize deoxynivalenol (DON), a mycotoxin, to deepoxy-deoxynivalenol (deepoxy-DON or DOM-1) or 3-epi-deoxynivalenol (3-epi-DON), respectively. Using different approaches, the intestinal toxicity of 3 molecules was compared and the molecular basis for the reduced toxicity investigated. In human intestinal epithelial cells, deepoxy-DON and 3-epi-DON were not cytotoxic, did not change the oxygen consumption or impair the barrier function. In intestinal explants, exposure for 4 hours to 10 µM DON induced intestinal lesions not seen in explants treated with deepoxy-DON and 3-epi-DON. A pan-genomic transcriptomic analysis was performed on intestinal explants. 747 probes, representing 323 genes, were differentially expressed, between DON-treated and control explants. By contrast, no differentially expressed genes were observed between control, deepoxy-DON and 3-epi-DON treated explants. Both DON and its biotransformation products were able to fit into the pockets of the A-site of the ribosome peptidyl transferase center. DON forms three hydrogen bonds with the A site and activates MAPKinases (mitogen-activated protein kinases). By contrast deepoxy-DON and 3-epi-DON only form two hydrogen bonds and do not activate MAPKinases. Our data demonstrate that bacterial de-epoxidation or epimerization of DON altered their interaction with the ribosome, leading to an absence of MAPKinase activation and a reduced toxicity.

Bacterial Epimerization as a Route for Deoxynivalenol Detoxification: the Influence of Growth and Environmental Conditions.

Deoxynivalenol (DON) is a toxic secondary metabolite produced by several Fusarium species that infest wheat and corn. Food and feed contaminated with DON pose a health risk to both humans and livestock and form a major barrier for international trade. Microbial detoxification represents an alternative approach to the physical and chemical detoxification methods of DON-contaminated grains. The present study details the characterization of a novel bacterium, Devosia mutans 17-2-E-8, that is capable of transforming DON to a non-toxic stereoisomer, 3-epi-deoxynivalenol under aerobic conditions, mild temperature (25-30°C), and neutral pH. The biotransformation takes place in the presence of rich sources of organic nitrogen and carbon without the need of DON to be the sole carbon source. The process is enzymatic in nature and endures a high detoxification capacity (3 µg DON/h/10(8) cells). The above conditions collectively suggest the possibility of utilizing the isolated bacterium as a feed treatment to address DON contamination under empirical field conditions.

Toxicology of 3-epi-deoxynivalenol, a deoxynivalenol-transformation product by Devosia mutans 17-2-E-8.

Microbial detoxification of deoxynivalenol (DON) represents a new approach to treating DON-contaminated grains. A bacterium Devosia mutans 17-2-E-8 was capable of completely transforming DON into a major product 3-epi-DON and a minor product 3-keto-DON. Evaluation of toxicities of these DON-transformation products is an important part of hazard characterization prior to commercialization of the biotransformation application. Cytotoxicities of the products were demonstrated by two assays: a MTT bioassay assessing cell viability and a BrdU assay assessing DNA synthesis. Compared with DON, the IC50 values of 3-epi-DON and 3-keto-DON were respectively 357 and 3.03 times higher in the MTT bioassay, and were respectively 1181 and 4.54 times higher in the BrdU bioassay. Toxicological effects of 14-day oral exposure of the B6C3F1 mouse to DON and 3-epi-DON were also investigated. Overall, there were no differences between the control (free of toxin) and the 25 mg/kg bw/day or 100 mg/kg bw/day 3-epi-DON treatments in body and organ weights, hematology and organ histopathology. However, in mice exposed to DON (2 mg/kg bw/day), white blood cell numbers and serum immunoglobulin levels were altered relative to controls, and lesions were observed in adrenals, thymus, stomach, spleen and colon. Taken together, in vitro and in vivo studies indicate that 3-epi-DON is substantially less toxic than DON.

An epimer of deoxynivalenol: purification and structure identification of 3-epi-deoxynivalenol.

In an investigation of deoxynivalenol (DON)-transformation products by Devosia mutans 17-2-E-8, the major product was identified as 3-epi-DON. This DON-transformation product was analysed by liquid chromatography and identified by congruent retention time and UV/Vis spectrum, as well as mass spectrometric data. Nuclear magnetic resonance (NMR) experiments including correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC) and nuclear overhauser effect (NOE) were conducted for structural characterisation of 3-epi-DON. High-speed counter-current chromatography (HSCCC) was applied to scale up the separation of 3-epi-DON from DON in a D. mutans 17-2-E-8 culture. From the culture where 100 mg DON was applied, 56 mg of 3-epi-DON (purity of 96.8%) was obtained from the HSCCC. The purified 3-epi-DON will be used for toxicological characterisation studies of this chemical.

[Observation on therapeutic effect of comprehensive therapy for acute large area cerebral infarction].

OBJECTIVE: To search for a method for increasing clinical therapeutic effect of acute large area cerebral infarction. METHODS: Ninety-six cases were randomly divided into a comprehensive treatment group and a western medicine group, 48 cases in each group. The western medicine group were treated with routine western methods, dehydration for decreasing intracranial pressure, brain-protection, anti-platelet agglutination, improving circulation and expectant treatment, etc.; the comprehensive treatment group were treated with the routine treatment of western medicine combined with acupuncture, moxibustion and enema of Chinese drugs. The therapeutic effect was observed after treatment for 28 days. RESULTS: The total effective rate was 68.8% in the comprehensive treatment group and 39.6% in the western medicine group with a very significant difference between the two groups (P < 0.01), and the score of the National Institute of Health's Stroke Scale (NIHSS) and the score of the Glasgow Coma Scale (GCS) in the comprehensive treatment group were better than those in the western medicine group (P < 0.001, P < 0.01). CONCLUSION: Combined treatment of Chinese medicine and western medicine for large area cerebral infarction is better than simple western medicine, and it can significantly improve neurological defect of the patient and decrease death rate.