Although widespread antibiotic resistance has been mostly attributed to the selective pressure generated by overuse and misuse of antibiotics, recent growing evidence suggests that chemicals other than antibiotics, such as certain metals, can also select and stimulate antibiotic resistance via both co-resistance and cross-resistance mechanisms. For instance, tetL, merE, and oprD genes are resistant to both antibiotics and metals. However, the potential de novo resistance induced by heavy metals at environmentally-relevant low concentrations (much below theminimum inhibitory concentrations [MICs], also referred as sub-inhibitory) has hardly been explored. This study investigated and revealed that heavy metals, namely Cu(II), Ag(I), Cr(VI), and Zn(II), at environmentally-relevant and sub-inhibitory concentrations, promoted conjugative transfer of antibiotic resistance genes (ARGs) between E. coli strains. The mechanisms of this phenomenon were further explored, which involved intracellular reactive oxygen species (ROS) formation, SOS response, increased cell membrane permeability, and altered expression of conjugation-relevant genes. These findings suggest that sub-inhibitory levels of heavy metals that widely present in various environments contribute to the resistance phenomena via facilitating horizontal transfer of ARGs. This study provides evidence from multiple aspects implicating the ecological effect of low levels of heavy metals on antibiotic resistance dissemination and highlights the urgency of strengthening efficacious policy and technology to control metal pollutants in the environments.
Endothelin receptor B subtype 2 (EDNRB2) is a seven-transmembrane G-protein coupled receptor. In this study, we investigated EDNRB2 gene as a candidate gene for duck spot plumage pattern according to studies of chicken and Japanese quail. The entire coding region was cloned by the reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis showed that duck EDNRB2 cDNA contained a 1311bp open reading frame and encoded a putative protein of 436 amino acids residues. The transcript shared 89%-90% identity with the counterparts in other avian species. A phylogenetic tree based on amino acid sequences showed that duck EDNRB2 was evolutionary conserved in avian clade. The entire coding region of EDNRB2 were sequenced in 20 spot and 20 non-spot ducks, and 13 SNPs were identified. Two of them (c.940G>A and c.995G>A) were non-synonymous substitutions, and were genotyped in 647 ducks representing non-spot and spot phenotypes. The c.995G>A mutation, which results in the amino acid substitution of Arg332His, was completely associated with the spot phenotype: all 152 spot ducks were carriers of the AA genotype and the other 495 individuals with non-spot phenotype were carriers of GA or GG genotype, respectively. Segregation in 17 GA×GG and 22 GA×GA testing combinations confirmed this association since the segregation ratios and genotypes of the offspring were in agreement with the hypothesis. In order to investigate the underlying mechanism of the spot phenotype, MITF gene was used as cell type marker of melanocyte progenitor cells while TYR and TYRP1 gene were used as cell type markers of mature melanocytes. Transcripts of MITF, TYR and TYRP1 gene with expected size were identified in all pigmented skin tissues while PCR products were not obtained from non-pigmented skin tissues. It was inferred that melanocytes are absent in non-pigmented skin tissues of spot ducks.
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