A Multiplex SYBR Green Real-Time PCR Assay for the Detection of Three Colistin Resistance Genes from Cultured Bacteria, Feces, and Environment Samples

The aim of the study was to develop a multiplex assay for rapid detection of mcr-1, mcr-2, and mcr-3, a group of genes of conferring resistance to colistin mediated by plasmid in Enterobacteriaceae. A SYBR Green based real-time PCR assay has been designed to detect the mcr genes, and applied to cultured bacteria, feces and soil samples. All three mcr genes could be detected with a lower limit of 102 cultured bacteria. This test was highly specific and sensitive, and generated no false-positive results. The assay was also conclusive when applied to feces and soil samples containing mcr-1-positive Escherichia coli, which could facilitate the screening of mcr genes not only in the bacteria, but also directly from the environment. This simple, rapid, sensitive, and specific multiplex assay will be useful for rapid screening of the colistin resistance in both clinical medicine and animal husbandry

Evaluation of the Toxicity and Toxicokinetics of Cereulide from an Emetic Bacillus cereus Strain of Milk Origin

Bacillus cereus is an opportunistic foodborne agent causing food poisoning and many infectious diseases. The heat-stable emetic toxin cereulide is one of the most prevalent toxins produced by pathogenic B. cereus, resulting in symptoms such as emesis and liver failure. In the present work, the toxicity and toxicokinetics of cereulide from an emetic B. cereus isolate (CAU45) of raw milk were evaluated. The production of cereulide was tested by a cytotoxicity test and enzyme immunoassay, and confirmed by the presence of the ces (cereulide synthetase) gene and the ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. All results showed that the amount and toxicity of cereulide produced by CAU45 was 7 to 15.3 folds higher than the reference emetic B. cereus DSMZ 4312. Cereulide in plasma was collected at different time points after a single intravenous injection to evaluate its toxicokinetics in rabbits. The maximum concentration of cereulide was achieved in 2.6 ± 3.4 h after administration, with the elimination half-life of 10.8 ± 9.1 h, which expands our understanding of the toxic effects of cereulide. Together, it suggests that urgent sanitary practices are needed to eliminate emetic toxins and emetic B. cereus in raw milk

Development of an indirect competitive ELISA based on immunomagnetic beads’ clean-up for detection of maduramicin in three chicken tissues

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on immunomagnetic beads' (IMBs) clean-up was developed for detection of the residues of maduramicin (MD) in different chicken tissues. IMBs coated with a specific monoclonal antibody (MAb) against MD named MAb 2D6 were applied to enrich the residues of MD in chicken tissues. The specificities of these IMBs were well maintained and the reversibility remained at more than 73% of the original capability after being used for three times. After elution, enriched MD was detected by a conventional ic-ELISA. The limits of detection of MD were 72, 74 and 173 μg/kg in chicken muscle, skin and fat, and liver, respectively. Recoveries ranged from 80.0% to 115.8% with coefficients of variation being less than 11.3%. These results indicated that a rapid, robust clean-up of IMBs combining ELISA provides a simple, time-saving and environmentally friendly method to detect MD in chicken tissues

RGAAT: A Reference-based Genome Assembly and Annotation Tool for New Genomes and Upgrade of Known Genomes

The rapid development of high-throughput sequencing technologies has led to a dramatic decrease in the money and time required for de novo genome sequencing or genome resequencing projects, with new genome sequences constantly released every week. Among such projects, the plethora of updated genome assemblies induces the requirement of version-dependent annotation files and other compatible public dataset for downstream analysis. To handle these tasks in an efficient manner, we developed the reference-based genome assembly and annotation tool (RGAAT), a flexible toolkit for resequencing-based consensus building and annotation update. RGAAT can detect sequence variants with comparable precision, specificity, and sensitivity to GATK and with higher precision and specificity than Freebayes and SAMtools on four DNA-seq datasets tested in this study. RGAAT can also identify sequence variants based on cross-cultivar or cross-version genomic alignments. Unlike GATK and SAMtools/BCFtools, RGAAT builds the consensus sequence by taking into account the true allele frequency. Finally, RGAAT generates a coordinate conversion file between the reference and query genomes using sequence variants and supports annotation file transfer. Compared to the rapid annotation transfer tool (RATT), RGAAT displays better performance characteristics for annotation transfer between different genome assemblies, strains, and species. In addition, RGAAT can be used for genome modification, genome comparison, and coordinate conversion. RGAAT is available at https://sourceforge.net/projects/rgaat/ and https://github.com/wushyer/RGAAT_v2 at no cost. Keywords: Variant identification, Genome assembly, Genome annotation, Genome compariso

Development of an indirect competitive ELISA based on immunomagnetic beads’ clean-up for detection of maduramicin in three chicken tissues

<p>An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on immunomagnetic beads' (IMBs) clean-up was developed for detection of the residues of maduramicin (MD) in different chicken tissues. IMBs coated with a specific monoclonal antibody (MAb) against MD named MAb 2D6 were applied to enrich the residues of MD in chicken tissues. The specificities of these IMBs were well maintained and the reversibility remained at more than 73% of the original capability after being used for three times. After elution, enriched MD was detected by a conventional ic-ELISA. The limits of detection of MD were 72, 74 and 173 μg/kg in chicken muscle, skin and fat, and liver, respectively. Recoveries ranged from 80.0% to 115.8% with coefficients of variation being less than 11.3%. These results indicated that a rapid, robust clean-up of IMBs combining ELISA provides a simple, time-saving and environmentally friendly method to detect MD in chicken tissues.</p

Presentation_1_Mucoid Acinetobacter baumannii enhances anti-phagocytosis through reducing C3b deposition.pdf

BackgroundMultidrug resistant (MDR) Acinetobacter baumannii causes serious infections in intensive care units and is hard to be eradicated by antibiotics. Many A. baumannii isolates are identified as the mucoid type recently, but the biological characteristics of mucoid A. baumannii and their interactions with host cells remains unclear.MethodsThe mucoid phenotype, antimicrobial susceptibility, biofilm-forming ability, acid resistance ability, peroxide tolerance, and in vivo toxicity of clinical ICUs derived A. baumannii isolates were first investigated. Secondly, the phagocytic resistance and invasive capacity of A. baumannii isolates to macrophages (MH-S, RAW264.7) and epithelial cells (A549) were analyzed. Furthermore, the abundance of C3b (complement factor C3 degradation product) deposition on the surface of A. baumannii was investigated. Last, the relationship between C3b deposition and the abundance of capsule in A. baumannii isolates were analyzed.ResultsThese A. baumannii strains showed different mucoid phenotypes including hyper mucoid (HM), medium mucoid (MM), and low mucoid (LM). All tested strains were MDR with high tolerance to either acid or hydrogen peroxide exposure. Notably, these mucoid strains showed the increase of mortality in the Galleria mellonella infection models. Besides, the HM strain exhibited less biofilm abundance, higher molecular weight (MW) of capsule, and greater anti-phagocytic activity to macrophages than the LM strain. Together with the increased abundance of capsule, high expression of tuf gene (associated with the hydrolysis of C3b), the HM strain effectively inhibits C3b deposition on bacterial surface, resulting in the low-opsonization phenotype.ConclusionCapsular characteristics facilitate the anti-phagocytic activity in hyper mucoid A. baumannii through the reduction of C3b deposition. Mucoid A. baumannii exhibits high phagocytosis resistance to both macrophages and epithelial cells.</p