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

Patients, healthcare providers, and general population preferences for hemodialysis vascular access: a discrete choice experiment

BackgroundA patient-centered dialysis treatment option requires an understanding of patient preferences for alternative vascular accesses and nephrologists often face difficulties when recommending vascular access to end-stage kidney disease (ESKD) patients. We aimed to quantify the relative importance of various vascular access characteristics to patients, healthcare providers and general population, and how they affect acceptability for patients and healthcare providers.MethodsIn a discrete choice experiment, patients with maintenance hemodialysis (MHD), healthcare providers, and individuals from the general population were invited to respond to a series of hypothetical vascular access scenarios that differed in five attributes: cumulative patency, infection rate, thrombosis rate, cost, and time to maturation. We estimated the respondents’ preference heterogeneity and relative importance of the attributes with a mixed logit model (MXL) and predicted the willingness to pay (WTP) of respondents via a multinomial logit model (MNL).ResultsHealthcare providers (n = 316) and the general population (n = 268) exhibited a favorable inclination toward longer cumulative patency, lower access infection rate and lower access thrombosis rate. In contrast, the patients (n = 253) showed a preference for a 3-year cumulative patency, 8% access infection rate, 35% access thrombosis rate and 1.5 access maturity time, with only the 3-year cumulative patency reaching statistical significance. Among the three respondent groups, the general population found cumulative patency less important than healthcare providers and patients did. Patients demonstrated the highest WTP for cumulative patency, indicating a willingness to pay an extra RMB$24,720(US$3,708) for each additional year of patency time.ConclusionPatients and healthcare providers had a strong preference for vascular access with superior patency. While the general population preferred vascular access with lower thrombosis rates. These results indicate that most patients prefer autogenous arteriovenous fistula (AVF) as an appropriate choice for vascular access due to its superior patency and lower complications than other vascular access types

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

Pseudorabies virus gM and its homologous proteins in herpesviruses induce mitochondria-related apoptosis involved in viral pathogenicity.

Apoptosis is a critical host antiviral defense mechanism. But many viruses have evolved multiple strategies to manipulate apoptosis and escape host antiviral immune responses. Herpesvirus infection regulated apoptosis; however, the underlying molecular mechanisms have not yet been fully elucidated. Hence, the present study aimed to study the relationship between herpesvirus infection and apoptosis in vitro and in vivo using the pseudorabies virus (PRV) as the model virus. We found that mitochondria-dependent apoptosis was induced by PRV gM, a late protein encoded by PRV UL10, a virulence-related gene involved in enhancing PRV pathogenicity. Mechanistically, gM competitively combines with BCL-XL to disrupt the BCL-XL-BAK complex, resulting in BCL-2-antagonistic killer (BAK) oligomerization and BCL-2-associated X (BAX) activation, which destroys the mitochondrial membrane potential and activates caspase-3/7 to trigger apoptosis. Interestingly, similar apoptotic mechanisms were observed in other herpesviruses (Herpes Simplex Virus-1 [HSV-1], human cytomegalovirus [HCMV], Equine herpesvirus-1 [EHV-1], and varicella-zoster virus [VZV]) driven by PRV gM homologs. Compared with their parental viruses, the pathogenicity of PRV-ΔUL10 or HSV-1-ΔUL10 in mice was reduced with lower apoptosis and viral replication, illustrating that UL10 is a key virulence-related gene in PRV and HSV-1. Consistently, caspase-3 deletion also diminished the replication and pathogenicity of PRV and HSV-1 in vitro and in mice, suggesting that caspase-3-mediated apoptosis is closely related to the replication and pathogenicity of PRV and HSV-1. Overall, our findings firstly reveal the mechanism by which PRV gM and its homologs in several herpesviruses regulate apoptosis to enhance the viral replication and pathogenicity, and the relationship between gM-mediated apoptosis and herpesvirus pathogenicity suggests a promising approach for developing attenuated live vaccines and therapy for herpesvirus-related diseases

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 <i>Bombyx mori singed</i> Gene Is Involved in the High-Temperature Resistance of Silkworms

Temperature is an important factor in the growth, development, survival, and reproduction of organisms. The high-temperature resistance mechanism of insects may be significant for use in the prevention and control of insect pests. The silkworm, Bombyx mori, is an important Lepidoptera model species for studies on pest control in agriculture and forestry. We identified a gene in B. mori, the B. mori singed (Bmsn) gene, which is involved in the high-temperature resistance of silkworms. Sn proteins are highly conserved among species in many taxonomic groups. The overexpression of the Bmsn gene promoted the proliferation of silkworm cells, reduced oxidation, and reduced the accumulation of reactive oxygen species under stress. Interfering with the Bmsn gene had the opposite result. We constructed a transgenic B. mori strain that overexpressed the Bmsn gene. The physiological traits of the transgenic strain were significantly improved, and it had stronger high-temperature resistance. The Bmsn gene is involved in the process by which fat bodies respond to high-temperature stress. These findings provide insights into the mechanism of high-temperature resistance of insects and offer a new perspective on agricultural and forestry pest control

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