Introduction: With the large-scale use of antibiotics, the detection rate and mortality of carbapenem resistant Escherichia coli (CR-EC) have gradually increased. This study investigated the molecular characteristics and prevalence of CR-EC in order to supplement the isolated data of CR-EC in Hangzhou, China. Methodology: The minimal inhibitory concentration was determined by microbroth dilution method. The drug resistance genes were detected by polymerase chain reaction. The transferability of plasmid was verified by the conjugation test and genetic homology was detected by pulsed-field gel electrophoresis. The whole genome was sequenced (WGS) using the Illumina MiSeq technology. Results: A total of 8 non-duplicated CR-EC isolates were collected, and all exhibited a multidrug-resistant phenotype. Two different New Delhi metallo-β-lactamase (NDM) variants, blaNDM-5 and blaNDM-13, were found with detection rates of 62.5% and 12.5%, respectively. The success rate of conjugation was 100% (6/6). Homology analysis showed that there was no widespread cloning outbreak of CR-EC, and blaNDM-5-ST410 was prevalent in the local area as a dominant group. WGS also indicated the rate of occurrence of resistance genes carrying resistance for more types of antibiotics, as well as exposed potential virulence risks. Conclusions: This was a survey on the prevalence and molecular characteristics of CR-EC in Hangzhou. blaNDM-like production combined with extended spectrum beta-lactamase (ESBLs) and/or AmpC was the main resistance mechanism of CR-EC in this area. The dominant blaNDM-5-ST410 requires enhanced attention. The horizontal transformation of plasmids, complex drug resistance, and potential virulence risks also need close attention.

Abstract Background: In recent years, with the large-scale use of antibiotics, the detection rate and mortality of carbapenem-resistant Escherichia coli (CR-EC) have gradually increased. This study explores the molecular characteristics and prevalence of CR-EC isolated from a tertiary hospital in Hangzhou. Methods: A total of 10 non-duplicated CR-EC were collected. The minimal inhibitory concentration was determined by microbroth dilution method. Various drug-resistant genes were detected by polymerase chain reaction. The transferability of plasmid was verified by conjugation test and genetic homology was detected by pulsed-field gel electrophoresis. The whole genome is sequenced using the Illumina MiSeq technology. Results: The strains were of diverse origin and all showed multidrug resistance. blaNDM was the only detected carbapenem gene with a high carrying rate (80%). The success rate of conjugation was 75%, indicating the horizontal transfer of blaNDM. Homology analysis showed that there was no widespread cloning outbreak of CR-EC in this study, blaNDM-5-ST410 is prevalent in the local area as a dominant group. WGS suggests complex plasmid replicon situations and greater potential risks of drug resistance and virulence. Conclusions: This is a survey on the prevalence and molecular characteristics of CR-EC in the Hangzhou, blaNDM-like production combined with ESBLs and/or AmpC was the main resistance mechanism of CR-EC. Complex drug resistance, potential virulence risks and the transferability of plasmids have intensified the trend of widespread prevalence of CR-EC, and medical personnel should closely monitor the changes of dominant clone groups (such as ST410) to prevent and control the further spread of CR-EC.

Cronobacter is an important foodborne pathogen that can cause severe neonatal meningitis, necrotizing enterocolitis, and bacteremia. Currently, there is limited knowledge of biofilm formation in Cronobacter. In the present study, biofilm formation ability and associated gene expression changes in Cronobacter from cereal related samples was carried out systematically. Our results from 307 Cronobacter isolates analyzed for 48 h showed strong biofilm-forming ability in 14 strains (4.6%), moderate in 47 strains (15.3%), weak in 142 strains (46.2%), and no such ability in the remaining 104 strains (33.9%). Further studies on five strains with strong biofilm-forming ability showed that maximum biofilm formation in Cronobacter occurred after 24 h of cultivation, reaching a peak around 48 h-72 h, reducing gradually thereafter. Kyoto encyclopedia of genes and genomes (KEGG) analysis revealed that differentially expressed genes (DEGs) involved in flagellar assembly, oxidative phosphorylation, ribosome, photosynthesis, O-Antigen nucleotide sugar biosynthesis, citrate cycle (tricarboxylic acid cycle, TCA) and bacterial chemotaxis were enriched in biofilm forming cells. The genes involved these enrichment pathways were mostly downregulated when compared to planktonic cells. Several transcriptional regulator genes such as csrA and bolA, and the cell surface composition regulator gene glgS were significantly upregulated. 12 of 13 (92.3%) selected genes was found to be in agreement with the RNA-Seq of planktonic and biofilm cells by Quantitative real-time PCR analysis, thus increasing confidence in our data. Our research lays a sound theoretical basis for further studies on mechanisms regulating biofilm formation and provides a foundation for development of new food safety measures, clinical disease prevention and control.

Introduction. The resistance rate of Klebsiella pneumoniae (K. pneumoniae) to imipenem is increasing year by year, and the imipenem resistance mechanism of K. pneumoniae is complex. Therefore, it is urgent to develop new strategies to explore the resistance mechanism of imipenem for its effective and accurate use in clinical practice.Hypothesis/Gap sStatement. Machine learning could identify resistance features and biological process that influence microbial resistance from whole-genome sequencing (WGS) data.Aims. This work aimed to predict imipenem resistance genetic features in K. pneumoniae from whole-genome k-mer features, and analyse their function for understanding its resistance mechanism.Methods. This study analysed WGS data of K. pneumoniae combined with resistance phenotype for imipenem, and established K. pneumoniae to imipenem genotype-phenotype model to predict resistance features using chi-squared test and random forest. An external clinical dataset was used to verify prediction power of resistance features. The potential genes were identified through alignment the resistance features with the K. pneumoniae reference genome using blastn, the functions of potential genes were further analysed to explore its resistance-related signalling pathways with GO and KEGG analysis, the resistance sequence patterns were screened using streme software. Finally, the resistance features were combined and modelled through four machine-learning algorithms (logistic regression, SVM, GBDT and XGBoost) to evaluate their phenotype prediction ability.Results. A total of 16 670 imipenem resistance features were predicted from genotype-phenotype model. The 30 potential genes were identified by annotating the resistance features and corresponded to known antibiotic-related genes (mdtM, dedA, rne, etc.). GO and KEGG pathway analyses indicated the possible association of imipenem resistance with metabolism process and cell membrane. CRYCAGCDN and CGRDAAAN were found from the imipenem resistance features, which were widely presented in the reported β-lactam resistance genes (bla SHV, bla CTX-M, bla TEM, etc.), and YCYAGCMCAST with metabolic functions (organic substance metabolic process, nitrogen compound metabolic process and cellular metabolic process) was identified from the top 50 resistance features. The 25 resistance genes in the training dataset included 19 genes in the external dataset, which verified the accuracy of prediction. The area under curve values of logistics regression, SVM, GBDT and XGBoost were 0.965, 0.966, 0.969 and 0.969, respectively, indicating that the imipenem resistance features have a strong prediction power.Conclusion. Machine-learning methods could effectively predict the imipenem resistance feature in K. pneumoniae, and provide resistance sequence profiles for predicting resistance phenotype and exploring potential resistance mechanisms. It provides an important insight into the potential therapeutic strategies of K. pneumoniae resistance to imipenem, and speed up the application of machine learning in routine diagnosis.

An oil well in an oil field had obvious deformation in the clamping area of the hydraulic clamp of the oil pipe during the string lifting of the wellbore dredging operation. In order to analyze the causes of tubing flattening, the author carried out macro morphology observation and analysis, geometric dimension measurement and nondestructive testing, and carried out chemical composition analysis, mechanical property test, metallographic structure analysis and energy spectrum analysis. The results show that the chemical composition, tensile properties and impact properties of the flattened tubing meet the standard requirements, and the metallographic structure is normal. The reason why the oil pipe is flattened is due to the excessive clamping force of the hydraulic tong during the lifting of the oil pipe. It is suggested to standardize the on-site tubing tripping operation and strengthen the product quality control of tubing entering the well. To prevent similar tubing failure from happening again.

Whole genome sequencing (WGS) of bacteria has become a routine method in diagnostic laboratories. One of the clinically most useful advantages of WGS is the ability to predict antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) in bacterial sequences. This allows comprehensive investigations of such genetic features but can also be used for epidemiological studies. A plethora of software programs have been developed for the detailed annotation of bacterial DNA sequences, such as rapid annotation using subsystem technology (RAST), Resfinder, ISfinder, INTEGRALL and The Transposon Registry. Unfortunately, to this day, a reliable annotation tool of the combination of ARGs and MGEs is not available, and the generation of genbank files requires much manual input. Here, we present a new webserver which allows the annotation of ARGs, integrons and transposable elements at the same time. The pipeline generates genbank files automatically, which are compatible with Easyfig for comparative genomic analysis. Our BacAnt code and standalone software package are available at https://github.com/xthua/bacant with an accompanying web application at http://bacant.net.

Vibrio parahaemolyticus is a major cause of seafood-associated food poisoning. It is of great significance to develop an accurate, simple and cost-effective method to identify infected seafood, especially for on-site application. Polymerase chain reaction (PCR) remains the golden standard for nucleic acid detection. But traditional methods heavily reply on sophisticated instrument and specialized operators, which limits the application for on-site detections. Here we developed a novel, specific and visualized detection method for PCR based on CRISPR/Cas12a system. On a low-cost thermal cycler, amplification reaction can be conducted easily. The CRISPR/Cas12a system was specifically designed to evaluate amplicons, eliminating false positive results. Besides the negative samples remained colorless, the positive samples generated obvious green fluorescence, which could be easily distinguished by the naked eye using a homemade UV device. The presented detection method was verified by detecting shrimp samples. The limit of detection is 1.02 × 102 copies/μL. This presented method provided a new strategy for specific endpoint detection of PCR and advanced its application in field for food safety assurance.

The microRNAs (miRNAs) are known to regulate immune functions in crustaceans, but little is known about the role of miRNAs against bacterial infection. We performed small RNA sequencing to characterize the differentially expressed microRNAs in V. parahemolyticus infected crab, in comparison to that in control uninfected crab, at 12 h and 24 h post infection. In total, 23 host miRNAs were up-regulated in response to the infection and 23 host miRNAs were down-regulated at both the time-points. Among these miRNAs, miR-125, PC-3p-11,483, miR-2815p, miR-10-3p and PC-5p-50,686 were significantly up-regulated by above 10 folds, and stem-loop RT-qPCR confirmed this result. Further, gene ontology analysis revealed that many signaling pathways, especially phagocytosis, were mediated by these miRNAs. Golgi apparatus and Magnesium ion binding are important biological processes, endocytosis and phagosome are important pathways in the immune response to V. parahemolyticus. This study is one important attempt at characterizing crab miRNAs that response to V. parahemolyticus infection, and will help unravel the miRNA pathways involved in antibacterial immunity of crab.

Currently, food allergy is a worldwide public health problem, of which peanut allergy is more severe. Peanut allergy is usually lifelong and even trace amount of peanut allergens can cause severe anaphylactic reactions. Nowadays there is no complete cure for sensitized individuals. The dominant prevention is to avoid exposure to peanuts and peanut products. But there are risks of false ingredient labeling and cross-contamination during food processing. Hence, it is important and urgent to detect peanut allergens in food products. This review highlights advances and future trends in DNA-based methods applied to detect peanut allergens in processed food. A summary of published methods for detecting peanut allergens in food is given with a comparison of DNA targets and limit of detection.

A quite simple, sensitive and visual detection method was developed forin situdetection ofV. parahaemolyticuswithin only 1 hour.