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Background Multidrug-resistant plasmids carrying replication genes have been widely present in various strains of Klebsiella pneumoniae. RepA and repB1 were found in plasmids belong to the IncFIB, but their detailed structural and genomic characterization was not reported yet. This is the first study that delivers structural and functional insights of repA- and repB1-carrying IncFIB plasmids. Methods Klebsiella pneumoniae strains A1705, 911021, and 1642 were isolated from the human urine samples and bronchoalveolar fluids collected from different hospitals of China. Antibacterial susceptibility and plasmid transfer ability were tested to characterize the resistant phenotypes mediated by the pA1705-qnrS, p911021-tetA, and p1642-tetA. The complete nucleotide sequences of these plasmids were determined through high-throughput sequencing technology and comparative genomic analyses of plasmids belong to the same incompatibility group were executed to extract the genomic variations and features. Results The pA1705-qnrS, p911021-tetA, and p1642-tetA are defined as non-conjugative plasmids, having two replication genes, repA and repB1 associated with IncFIB family, and unknown incompatible group, respectively. Comparative genomic analysis revealed that relatively small backbones of IncFIB plasmids integrated massive accessory module at one "hotspot" that was located between orf312 and repB1. These IncFIB plasmids exhibited the distinct profiles of accessory modules including one or two multidrug-resistant regions, many complete and remnant mobile elements comprising integrons, transposons and insertion sequences. The clusters of resistant genes were recognized in this study against different classes of antibiotics including β-lactam, phenicol, aminoglycoside, tetracycline, quinolone, trimethoprim, sulfonamide, tunicamycin, and macrolide. It has been observed that all resistant genes were located in multidrug resistance regions. Conclusion It is concluded that multidrug-resistant repA and repB1-carrying IncFIB plasmids are a key source to mediate the resistance through mobile elements among Klebsiella pneumoniae. Current findings provide a deep understanding of horizontal gene transfer among plasmids of the IncFIB family via mobile elements that will be utilized in further in vitro studies.

Pharmaceuticals and personal care products (PPCPs) as emerging contaminants are ubiquitously present in the aquatic environment. Using in vivo and in silico techniques, this study aims to elucidate tissue distribution and endocrine disruption effects of chronic exposure (120 days) to PPCP mixture at environmentally relevant concentrations (ERCs) in adult zebrafish. Results from UHPLC-MS/MS analyses showed elevated distribution of PPCPs in zebrafish tissues in the order of liver > gonad > brain. Upregulation of steroid hormone receptors, both gonadotropin, and steroidogenic genes perturb the HPG axis pathway in females, while male fish exhibited significantly downregulated expressions of vtg, cyp17, and 17βhsd genes with inhibited fecundity. The Spearman correlation indicated a significant positive relationship between PPCPs bioaccumulation and mRNA levels of HPG axis genes. In silico molecular docking (MD) revealed specific amino acid residues of PPCPs binding with zebrafish estrogen receptors. Furthermore, the strongest binding energies of sulfamethoxazole, carbamazepine, and triclosan were discovered in erα and erβ estrogen receptors, confirming PPCPs' xenoestrogenic behavior. To summarize, chronic exposure to ERCs resulted in a high accumulation of PPCPs in the liver and gonad tissues of adult zebrafish, as well as associated perturbed genetic responses. As a result, strict environmental regulations for the disposal of PPCPs should be ensured to protect ecological and public health.

Di-(2-ethylhexyl) phthalate (DEHP) can affect the male reproductive system in vertebrates, but the underlying molecular mechanism is still elusive. Therefore, in this study, we aimed to dig the in-depth mechanism of DEHP-induced reproductive toxicity on male zebrafish via testicular transcriptome using embryo exposed at the environmentally relevant concentration (ERC) of 100 μg/L for 111 days. Moreover, our results were further confirmed via in silico technique and bioassay experimental in vitro (cell lines) and in vivo (zebrafish). The results showed DEHP exposure could affect male spermatogenesis, altered gonad histology, and reduced egg fertilization rate. Transcriptome analysis identified 1879 significant differentially expressed genes enriched in the exposure group. Twenty-seven genes related to three pathways of reproduction behavior were further validated by qPCR. In silico molecular docking revealed that DEHP and its metabolism bind to the zebrafish progesterone receptor (Pgr), suggesting the potential disruption of DEHP to the normal Pgr signaling. To further validate it, a wild-type Pgr plasmid and its mutants on specific binding sites were constructed. The transfection and microinjection experiment demonstrated that these binding sites mutations of Pgr affected the expression levels of male reproductive toxicity. Taken together, our study provided new insight into the molecular mechanisms of male reproductive toxicity induced by DEHP, and Pgr may serve as an important target binding by DEHP pollution, which needs further study in the future.

Phthalate esters (PAEs) pose prominent ecological risks owing to their multiplex toxicity potentials and ubiquitous detection in the environment. Therefore, this study aims to prioritize the individual and mixtures of six PAEs based on their toxicological implications using in vitro and vivo models exposed at environmentally relevant concentrations. Results were further confirmed using in silico Combination index (CI) and Independent action (IA), and molecular docking models. Among PAEs, DEHP revealed prominent in vitro/vivo toxicity followed by DEP, DBP, and DMP. Importantly, binary mixtures particularly C2-C6 and C11-C15 exhibited greater developmental toxicity, apoptosis, and perturbed the HPG pathway. The CI and IA models forecasted antagonistic and additive effects at Fa = 0.5 and Fa = 0.9 using in vitro Acinetobacter sp. Tox2. Conversely, in zebrafish, the IA model predicted mixture effects in the following order: additive > synergistic > antagonistic on the regulation of the HPG pathway, which was consistent with experimental results from Acridine Orange (AO) staining and apoptosis gene expression. Molecular docking for estrogen receptors (ERα, ERβ) revealed the highest binding energy scores for DEHP, compared to other PAEs. In short, our findings confirm that individual and mixtures of PAEs behave as xenoestrogens in the freshwater ecosystem with DEHP as a priority compound.

Background Multidrug-resistant plasmids carrying replication genes have been widely present in various strains of Klebsiella pneumoniae. RepA and repB1 were found in plasmids belong to the IncFIB, but their detailed structural and genomic characterization was not reported yet. This is the first study that delivers structural and functional insights of repA- and repB1-carrying IncFIB plasmids. Methods Klebsiella pneumoniae strains A1705, 911021, and 1642 were isolated from the human urine samples and bronchoalveolar fluids collected from different hospitals of China. Antibacterial susceptibility and plasmid transfer ability were tested to characterize the resistant phenotypes mediated by the pA1705-qnrS, p911021-tetA, and p1642-tetA. The complete nucleotide sequences of these plasmids were determined through high-throughput sequencing technology and comparative genomic analyses of plasmids belong to the same incompatibility group were executed to extract the genomic variations and features. Results The pA1705-qnrS, p911021-tetA, and p1642-tetA are defined as non-conjugative plasmids, having two replication genes, repA and repB1 associated with IncFIB family, and unknown incompatible group, respectively. Comparative genomic analysis revealed that relatively small backbones of IncFIB plasmids integrated massive accessory module at one “hotspot” that was located between orf312 and repB1. These IncFIB plasmids exhibited the distinct profiles of accessory modules including one or two multidrug-resistant regions, many complete and remnant mobile elements comprising integrons, transposons and insertion sequences. The clusters of resistant genes were recognized in this study against different classes of antibiotics including β-lactam, phenicol, aminoglycoside, tetracycline, quinolone, trimethoprim, sulfonamide, tunicamycin, and macrolide. It has been observed that all resistant genes were located in multidrug resistance regions. Conclusion It is concluded that multidrug-resistant repA and repB1-carrying IncFIB plasmids are a key source to mediate the resistance through mobile elements among Klebsiella pneumoniae. Current findings provide a deep understanding of horizontal gene transfer among plasmids of the IncFIB family via mobile elements that will be utilized in further in vitro studies.