The propagation of antibiotic resistance genes (ARGs) represents a global threat to both human health and food security. Assessment of ARG reservoirs and persistence is therefore critical for devising and evaluating strategies to mitigate ARG propagation. This study developed a novel, internal standard method to extract extracellular DNA (eDNA) and intracellular DNA (iDNA) from water and sediments, and applied it to determine the partitioning of ARGs in the Haihe River basin in China, which drains an area of intensive antibiotic use. The concentration of eDNA was higher than iDNA in sediment samples, likely due to the enhanced persistence of eDNA when associated with clay particles and organic matter. Concentrations of sul1, sul2, tetW, and tetT antibiotic resistance genes were significantly higher in sediment than in water, and were present at higher concentrations as eDNA than as iDNA in sediment. Whereas ARGs (frequently located on plasmid DNA) were detected for over 20 weeks, chromosomally encoded 16S rRNA genes were undetectable after 8 weeks, suggesting higher persistence of plasmid-borne ARGs in river sediment. Transformation of indigenous bacteria with added extracellular ARG (i.e., kanamycin resistance genes) was also observed. Therefore, this study shows that extracellular DNA in sediment is a major ARG reservoir that could facilitate antibiotic resistance propagation.

A competitive indirect enzyme-linked immunosorbent assay (ciELISA) using monoclonal antibodies (Mabs) having broad specificity for fluoroquinolone (FQ) antibiotics is described. Four FQs, ciprofloxacin (CIP), enrofloxacin (ENR), norfloxacin (NOR), and ofloxacin (OFL), were conjugated to bovine serum albumin for immunogens and to ovalbumin for coating antigens. A Mab C4A9H1 raised against the CIP hapten exhibited high cross-reactivity (35-100%) with 12 of 14 FQs and detected these FQs in a ciELISA below their maximum residue levels (MRLs) with good sensitivity at 50% binding inhibition (IC50). The quantitative structure-activity relationship (QSAR) between Mab C4A9H1 and various FQs by comparative molecular field analysis (CoMFA) showed a high predictive ability with a cross-validation q2 value of 0.866. Using a simple purification process and the broad-specificity ciELISA adapted for analysis of FQs in chicken muscle, chicken liver, honey, shrimp, and whole egg samples demonstrated recoveries of 60-93% for CIP, ENR, NOR, OFL, flumequine, and danofloxacin.

BackgroundThe 5’ region of cytochrome oxidase I (COI) is the standard marker for DNA barcoding. However, COI has proved to be of limited use in identifying some species, and for some taxa, the coding sequence is not efficiently amplified by PCR. These deficiencies lead to uncertainty as to whether COI is the most suitable barcoding fragment for species identification of ticks.MethodsIn this study, we directly compared the relative effectiveness of COI, 16S ribosomal DNA (rDNA), nuclear ribosomal internal transcribed spacer 2 (ITS2) and 12S rDNA for tick species identification. A total of 307 sequences from 84 specimens representing eight tick species were acquired by PCR. Besides the 1,834 published sequences of 189 tick species from GenBank and the Barcode of Life Database, 430 unpublished sequences representing 59 tick species were also successfully screened by Bayesian analyses. Thereafter, the performance of the four DNA markers to identify tick species was evaluated by identification success rates given by these markers using nearest neighbour (NN), BLASTn, liberal tree-based or liberal tree-based (+threshold) methods.ResultsGenetic divergence analyses showed that the intra-specific divergence of each marker was much lower than the inter-specific divergence. Our results indicated that the rates of correct sequence identification for all four markers (COI, 16S rDNA, ITS2, 12S rDNA) were very high (> 96%) when using the NN methodology. We also found that COI was not significantly better than the other markers in terms of its rate of correct sequence identification. Overall, BLASTn and NN methods produced higher rates of correct species identification than that produced by the liberal tree-based methods (+threshold or otherwise).ConclusionsAs the standard DNA barcode, COI should be the first choice for tick species identification, while 16S rDNA, ITS2, and 12S rDNA could be used when COI does not produce reliable results. Besides, NN and BLASTn are efficient methods for species identification of ticks.

4-Coumaric acid:CoA ligase (4CL) is the central enzyme of the plant-specific phenylpropanoid pathway. It catalyzes the synthesis of hydroxycinnamate-CoA thioesters, the precursors of lignin and other important phenylpropanoids, in two-step reactions involving the formation of hydroxycinnamate-AMP anhydride and then the nucleophilic substitution of AMP by CoA. In this study, we determined the crystal structures of Populus tomentosa 4CL1 in the unmodified (apo) form and in forms complexed with AMP and adenosine 59-(3-(4-hydroxyphenyl)propyl)phosphate (APP), an intermediate analog, at 2.4, 2.5, and 1.9 Å resolution, respectively. 4CL1 consists of two globular domains connected by a flexible linker region. The larger N-domain contains a substrate binding pocket, while the C-domain contains catalytic residues. Upon binding of APP, the C-domain rotates 818 relative to the N-domain. The crystal structure of 4CL1-APP reveals its substrate binding pocket. We identified residues essential for catalytic activities (Lys-438, Gln-443, and Lys-523) and substrate binding (Tyr-236, Gly-306, Gly-331, Pro-337, and Val-338) based on their crystal structures and by means of mutagenesis and enzymatic activity studies. We also demonstrated that the size of the binding pocket is the most important factor in determining the substrate specificities of 4CL1. These findings shed light on the enzymatic mechanisms of 4CLs and provide a solid foundation for the bioengineering of these enzymes.

The global increase in multidrug resistant (MDR) bacteria has led to phage therapy being refocused upon. A novel endolysin, LysPA26, containing a lysozyme-like domain, was screened against Pseudomonas aeruginosa in this study. It had activity against MDR P. aeruginosa without pretreatment with an outer-membrane permeabilizer. LysPA26 could kill up to 4 log units P. aeruginosa in 30 min. In addition, temperature and pH effect assays revealed that LysPA26 had good stability over a broad range of pH and temperatures. Moreover, LysPA26 could kill other Gram-negative bacteria, such as Klebsiella pneumonia, Acinetobacter baumannii and Escherichia coli, but not Gram-positive bacteria. Furthermore, LysPA26 could eliminate P. aeruginosa in biofilm formation. Our current results show that LysPA26 is a new and promising antimicrobial agent for the combat of Gram-negative pathogens.