Nucleotide sequences of 16 transmissible plasmids identified in nine multidrug-resistant Escherichia coli isolates expressing an ESBL phenotype isolated from food-producing animals and healthy humans

Objectives Nine extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolated from healthy humans and food-producing animals were found to transfer their cefotaxime resistance marker at high frequency in laboratory conjugation experiments. The objective of this study was to completely characterize 16 transmissible plasmids that were detected in these bacterial isolates. Methods The nucleotide sequences of all 16 plasmids were determined from transconjugants using next-generation sequencing technology. Open reading frames were assigned using Rapid Annotation using Subsystem Technology and analysed by BLASTn and BLASTp. The standard method was used for plasmid multilocus sequence typing (pMLST) analysis. Plasmid structures were subsequently confirmed by PCR amplification of selected regions. Results The complete circularized nucleotide sequence of 14 plasmids was determined, along with that of a further two plasmids that could not be confirmed as closed. These ranged in size from 1.8 to 166.6 kb. Incompatibility groups and pMLSTs identified included IncI1/ST3, IncI1/ST36, IncN/ST1, IncF and IncB/O, and those of the same Inc types presented a similar backbone structure despite being isolated from different sources. Eight plasmids contained blaCTX-M-1 genes that were associated with either ISEcp1 or IS26 insertion sequence elements. Six plasmids isolated from humans and chickens were identical or closely related to the IncI1 reference plasmid, R64. Conclusions These data, based on comparative sequence analysis, highlight the successful spread of blaESBL-harbouring plasmids of different Inc types among isolates of human and food-producing animal origin and provide further evidence for potential dissemination route

Surveillance, survival and adaptation of Cronobacter species in low-moisture environments

Cronobacter species (formerly known as Enterobacter sakazakii) are opportunistic pathogens that consist of seven species including C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis, C. dublinensis, C. universalis, and C. condimenti. This bacterium can cause meningitis, necrotizing enterocolitis, bacteraemia and sepsis, predominantly in neonates with an under developed immune system, following the consumption of contaminated powdered infant formula (PIF). Therefore, Cronobacter represents an important challenge for the PIF industry.The reference method for the detection of Cronobacter takes up to seven days for the confirmation by using conventional bacteriological culture. Thus a detection platform, which can provide a reliable result within a shorter time frame, would be desirable for the PIF industry. Such a platform, known as Vitek Immuno Diagnostic Assay System (VIDAS®), was investigated for its specificity, sensitivity and accuracy, when Cronobacter was present in TSB culture or reconstituted PIF, or in the presence of other non-related competing species. Overall, VIDAS® Cronobacter kits can effectively detect most Cronobacter species within a 20-h period. This approach may provide a useful basis upon which to improve positive release protocols for the PIF industry generally.Serotyping often plays an essential role in the identification of bacteria of importance to human health. This strategy is based on the detection of cell surface antigens, thereby facilitating the epidemiologic classification of bacteria to the sub-species level. Previously our laboratory described the first two major serotypes for Cronobacter sakazakii, denoted as O:1 and O:2. Efforts in developing new serotyping protocols continue to be reported. In this study, five unique serotypes were identified, including C. turicensis O:3, C. muytjensii O:2, C. dublinensis O:1, C. dublinensis O:2, and C. universalis O:1.Monitoring the microbial ecology of PIF production sites is an important step in an attempt to limit the risk of contamination in the finished food products. Cronobacter species, like other microorganisms can adapt to the production environment. To date sequence type 4 (ST-4) has been linked with recorded cases of meningitis, which have been isolated from PIF and its production environment. In this thesis, we reported on a 26-month surveillance study performed in an effort to identify and characterise persistent ST type(s), cultured from four PIF production facilities, using molecular strategies including target-specific polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST) and multi-genome microarray. Phenotypes traits including bacterial motility, biofilm formation, as well as morphotypes, were further invesigated on all these isolates. These phenotypes were considered to be among the most relevant to support bacteria in such harsh environments. Results showed that C. sakazakii serotype O:1, ST-1 was the most commonly recognised sequence type in PIF and its production environment. Significant differences were noted based on the phenotypes expressed by ST-1 and -4 isolates. ST-1 isolates cultured from PIF, formed a stronger biofilm at both 28 and 37°C when compared to ST-4 of clinical origin; while the latter exhibited a higher swim activity and an increased Congo red dye binding. This may represent a form of patho-adaptation. Understanding how this pathogen adapts to the PIF production environment will support targeted improvements in food safety measures.Cronobacter species is known for its desiccation tolerance, a phenotype that can aid bacterial survival in the production site and in PIF itself. In this study the complete genome sequence of one such isolate found to persist in a PIF production facility, denoted as C. sakazakii SP291, along with its phenotypic characteristics were reported. The genome of C. sakazakii SP291 consists of a 4.3 Mb chromosome (56.9% GC) along with three plasmids, denoted as pSP291-1 (118.1 kb, 57.2% GC), pSP291-2 (52.1 kb, 49.2% GC) and pSP291-3 (4.4 kb, 54.0% GC). When compared with C. sakazakii ATCC® BAA-894, which is also of PIF origin, the annotated genome sequence identified two interesting functional categories, comprising of genes related to the bacterial stress response along with resistance to antimicrobial and toxic compounds. Using phenotypic microarray (PM), a full metabolic profile was provided comparing C. sakazakii SP291 with C. sakazakii ATCC® BAA-894. These data extend our understanding of the genome of this important neonatal pathogen and provide further insights into the genotypes associated with features that can contribute to its persistence in the PIF environments.Little is known about the mechanisms that Cronobacter species deploy to survive and persist in low-moisture environments, including the PIF production environment. The aim of this study was to explore the gene signalling contributing to the survival and persistence phenotype in low-moisture environments by using the well-characterised persistent PIF environmental isolate, C. sakazakii SP291. The gene expression profiles related to desiccation were investigated using RNA sequencing (RNA-seq). Overall the most up- regulated genes were identified and found to be involved in the osmotic stress response. These included the ProU system (composed of proV, proX, and proW) and the bet-encoding operon (betIAB), all of which have been reported previously in other microorganisms including E. coli and Salmonella species. Interestingly four stress response genes were involved with down-regulation, in particular yehW, a gene known to play a role in the osmoprotectant uptake system of E. coli. Our observations from RNA-seq were validated using reverse transcription PCR (RT-PCR) with a selected sub-set of these gene targets.Meanwhile a transposon-mutant library was constructed in C. sakazakii SP291 separately. Pools of random insertion mutants were similarly desiccated (as for the RNA-seq experiment) following series of passages in bacterial culture. The mutant library was screened by transposon-directed insertion site sequencing (TraDIS) and compared against the original, to identify those genes that are required to support survival in low-moisture conditions. TraDIS identified 258 genes required for fundamental biological processes, 133 advantageous genes for growth under standard laboratory conditions as well as 43 advantageous genes required for growth at the defined environmental conditions. Some 32 genes have significant fold-changes after desiccation as assayed by TraDIS. Comparing the data outputs from TraDIS with RNA-seq, 11 genes were mapping by RNA-seq and defined as being up-regulated, and 2 genes being greatly down-regulated. In conclusion, this study used two high-throughput technologies to investigate the gene signalling and requirements in Cronobacter sakazakii when exposed to low-moisture conditions. Together these findings highlight some of the important biomarkers that need to be further assessed for their roles in this process.In general data from these studies, will contribute to the future development of improved detection and identification strategies for this important neonatal pathogen. Furthermore the RNA-seq and TraDIS experiments will provide early insights that can be a solid foundation for later studies, to extend our understanding of how this bacterium remains viable in a low-moisture food matrix and the environment in which it is often found. In the future these data will contribute to reduce the risk of Cronobacter contamination in PIF and its production environments, thereby improving food safety and protecting public health.2 year embargo added by autho

Nontargeted metabolomics analysis of follicular fluid in patients with endometriosis provides a new direction for the study of oocyte quality

Abstract Endometriosis is a common, estrogen‐dependent chronic gynecological disease that endangers the reproductive system and systemic metabolism of patients. We aimed to investigate the differences in metabolic profiles in the follicular fluid between infertile patients with endometriosis and controls. A total of 25 infertile patients with endometriosis and 25 infertile controls who were similar in age, BMI, fertilization method and ovulation induction treatment were recruited in this study. Metabolomics analysis of follicular fluid was performed by two methods of high‐performance liquid chromatography tandem mass spectrometry. There were 36 upregulated and 17 downregulated metabolites in the follicular fluid of patients in the endometriosis group. KEGG pathway analysis revealed that these metabolites were enriched in phenylalanine, tyrosine and tryptophan biosynthesis, aminoacyl‐tRNA biosynthesis, phenylalanine metabolism and pyrimidine metabolism pathways. A biomarker panel consisting of 20 metabolites was constructed by random forest, with an accuracy of 0.946 and an AUC of 0.988. This study characterizes differences in follicular fluid metabolites and associated pathway profiles in infertile patients with endometriosis. These findings can provide a better comprehensive understanding of the disease and a new direction for the study of oocyte quality, as well as potential metabolic markers for the prognosis of endometriosis

Comparative genotypic and phenotypic analysis of Cronobacter species cultured from four powdered infant formula production facilities: indication of pathoadaptation along the food chain

Cronobacter species are opportunistic pathogens commonly found in the environment. Among the seven Cronobacter species, Cronobacter sakazakii sequence type 4 (ST-4) is predominantly associated with recorded cases of infantile meningitis. This study reports on a 26-month powdered infant formula (PIF) surveillance program in four production facilities located in distinct geographic regions. The objective was to identify the ST(s) in PIF production environments and to investigate the phenotypic features that support their survival. Of all 168 Cronobacter isolates, 133 were recovered from a PIF production environment, 31 were of clinical origin, and 4 were laboratory type strains. Sequence type 1 (n = 84 isolates; 63.9%) was the dominant type in PIF production environments. The majority of these isolates clustered with an indistinguishable pulsotype and persisted for at least an 18-month period. Moreover, DNA microarray results identified two phylogenetic lineages among ST-4 strains tested. Thereafter, the ST-1 and -4 isolates were phenotypically compared. Differences were noted based on the phenotypes expressed by these isolates. The ST-1 PIF isolates produced stronger biofilms at both 28°C and 37°C, while the ST-4 clinical isolates exhibited greater swimming activity and increased binding to Congo red dye. Given the fact that PIF is a low-moisture environment and that the clinical environment provides for an interaction between the pathogen and its host, these differences may be consistent with a form of pathoadaptation. These findings help to extend our current understanding of the epidemiology and ecology of Cronobacter species in PIF production environment

The TNNI3 p.R186Q mutation is responsible for hypertrophic cardiomyopathy via promoting FASN-stimulated abnormal fatty acid metabolism

Introduction: The TNNI3 gene encodes the protein of cardiac troponin I (cTnI), which is an inhibitory subunit of sarcomeres. Mutations in this gene account for 3% of hypertrophic cardiomyopathy (HCM) and the molecular mechanism is complex. Recently, lipid metabolism has been revealed to be involved in HCM. Aim: The purpose of this work is to identify whether the pathological mechanism of the hotspot mutation TNNI3 p.R186Q in HCM is related to abnormal lipid metabolism.Methods and Results: A knock-in (KI) mouse model carrying the Tnni3 p.R186Q homozygous mutation (Tnni3R186Q/R186Q) was novelty generated by CRISPR/Cas9 technology and successfully constructed a typical phenotype of cardiac-myopathy. Likewise, neonatal rat cardiomyocytes (NRCMs) transfected with a mutant plasmid with the TNNI3 p.R186Q mutation showed the same phenomenon. In-depth experiments on related functions and molecular mechanisms were conducted, and Tnni3R186Q/R186Q mice exhibited abnormal fatty acid metabolism, which was induced by the activation of epidermal growth factor receptor (EGFR)-dependent high expression of fatty acid synthase (FASN) in vivo and in vitro. Specifically, the direct binding of EGFR and cTnI was destroyed by TNNI3 p.R186Q mutation, as observed through bioinformatics, Co-IP and GST-pull down analysis.Conclusion: In the present study, we successfully engineered Tnni3R186Q/R186Q mice with the typical phenotype of myocardial hypertrophy. We demonstrated that the TNNI3 p.R186Q mutation could induce HCM by the dissociation of EGFR and cTnI, which further led to EGFR-dependent increased expression of FASN and abnormal lipid metabolism

Variation of DNA Methylome of Zebrafish Cells under Cold Pressure

<div><p>DNA methylation is an essential epigenetic mechanism involved in multiple biological processes. However, the relationship between DNA methylation and cold acclimation remains poorly understood. In this study, Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) was performed to reveal a genome-wide methylation profile of zebrafish (Danio rerio) embryonic fibroblast cells (ZF4) and its variation under cold pressure. MeDIP-seq assay was conducted with ZF4 cells cultured at appropriate temperature of 28°C and at low temperature of 18°C for 5 (short-term) and 30 (long-term) days, respectively. Our data showed that DNA methylation level of whole genome increased after a short-term cold exposure and decreased after a long-term cold exposure. It is interesting that metabolism of folate pathway is significantly hypomethylated after short-term cold exposure, which is consistent with the increased DNA methylation level. 21% of methylation peaks were significantly altered after cold treatment. About 8% of altered DNA methylation peaks are located in promoter regions, while the majority of them are located in non-coding regions. Methylation of genes involved in multiple cold responsive biological processes were significantly affected, such as anti-oxidant system, apoptosis, development, chromatin modifying and immune system suggesting that those processes are responsive to cold stress through regulation of DNA methylation. Our data indicate the involvement of DNA methylation in cellular response to cold pressure, and put a new insight into the genome-wide epigenetic regulation under cold pressure.</p></div

RNA sequencing-based transcriptional overview of Xerotolerance in Cronobacter sakazakii SP291

Cronobacter sakazakii is a xerotolerant neonatal pathogen epidemiologically linked to powdered infant food formula, often resulting in high mortality rates. Here, we used transcriptome sequencing (RNA-seq) to provide transcriptional insights into the survival of C. sakazakii in desiccated conditions. Our RNA-seq data show that about 22% of the total C. sakazakii genes were significantly upregulated and 9% were downregulated during desiccation survival. When reverse transcription-quantitative PCR (qRT-PCR) was used to validate the RNA-seq data, we found that the primary desiccation response was gradually downregulated during the tested 4 hours of desiccation, while the secondary response remained constitutively upregulated. The 4-hour desiccation tolerance of C. sakazakii was dependent on the immediate microenvironment surrounding the bacterial cell. The removal of Trypticase soy broth (TSB) salts and the introduction of sterile infant formula residues in the microenvironment enhanced the desiccation survival of C. sakazakii SP291. The trehalose biosynthetic pathway encoded by otsA and otsB, a prominent secondary bacterial desiccation response, was highly upregulated in desiccated C. sakazakii. C. sakazakii SP291 ΔotsAB was significantly inhibited compared with the isogenic wild type in an 8-hour desiccation survival assay, confirming the physiological importance of trehalose in desiccation survival. Overall, we provide a comprehensive RNA-seq-based transcriptional overview along with confirmation of the phenotypic importance of trehalose metabolism in Cronobacter sakazakii during desiccation. IMPORTANCE Cronobacter sakazakii is a pathogen of importance to neonatal health and is known to persist in dry food matrices, such as powdered infant formula (PIF) and its associated production environment. When infections are reported in neonates, mortality rates can be high. The success of this bacterium in surviving these low-moisture environments suggests that Cronobacter species can respond to a variety of environmental signals. Therefore, understanding those signals that aid the persistence of this pathogen in these ecological niches is an important step toward the development of strategies to reduce the risk of contamination of PIF. This research led to the identification of candidate genes that play a role in the persistence of this pathogen in desiccated conditions and, thereby, serve as a model target to design future strategies to mitigate PIF-associated survival of C. sakazakii

Claudin18.2 bispecific T cell engager armed oncolytic virus enhances antitumor effects against pancreatic cancer

Bispecific T cell engagers (BiTEs) represent a promising immunotherapy, but their efficacy against immunologically cold tumors such as pancreatic ductal adenocarcinoma remains unclear. Oncolytic viruses (OVs) can transform the immunosuppressive tumor microenvironment into the active state and also serve as transgene vectors to selectively express the desired genes in tumor cells. This study aimed to investigate whether the therapeutic benefits of tumor-targeting Claudin18.2 BiTE can be augmented by combining cancer selectively and immune-potentiating effects of OVs. Claudin18.2/CD3 BiTE was inserted into herpes simplex virus type 1 (HSV-1) to construct an OV-BiTE. Its expression and function were assessed using reporter cells and peripheral blood mononuclear cell (PBMC) co-culture assays. Intratumoral application of OV-BiTE restrained tumor growth and prolonged mouse survival compared with the unarmed OV in xenograft models and syngeneic mice bearing CLDN18.2-expressing KPC or Pan02 pancreatic cancer cells. Flow cytometry of tumor-infiltrating immune cells suggested both OV-BiTE and the unarmed OV remodeled the tumor microenvironment by increasing CD4+ T cell infiltration and decreasing regulatory T cells. OV-BiTE further reprogrammed macrophages to a more pro-inflammatory antitumor state, and OV-BiTE-induced macrophages exhibited greater cytotoxicity on the co-cultured tumor cell. This dual cytotoxic and immunomodulatory approach warrants further development for pancreatic cancer before clinical investigation