tet(X4) is the critical resistance gene for tigecycline degradation that has been continually reported in recent years. In particular, pathogenic bacteria carrying tet(X4) are a severe threat to human health. However, information describing Escherichia coli coharboring tet(X4) with virulence genes is limited. Here, we isolated an E. coli strain coharboring tet(X4) and the heat-stable toxin gene astA from a dead piglet. The strain named 812A1-131 belongs to ST10. The genome was sequenced using the Nanopore and Illumina platforms. The virulence genes astA and tet(X4) are located on the chromosome and in the IncHI1-type plasmid p812A1-tetX4-193K, respectively. The plasmid could be conjugatively transferred to recipient E. coli J53 with high frequency. In vivo experiments showed that strain 812A1-131 is pathogenic to Galleria mellonella and could colonize the intestines of mice. In summary, pathogenic E. coli could receive a plasmid harboring the tet(X4) gene, which can increase the difficulty of treatment. The prevalence and transmission mechanisms of pathogenic bacteria coharboring the tet(X4) gene need more attention.

High temperatures exacerbate the ammonia inhibition of anaerobic digestion coupled with methanogenesis. The inhibition of methane production by ammonia has been observed in other studies. However, the underlying mechanism is not well understood and requires further investigation. This study explored the effect of ammonia stress on archaeal 16S rRNA transcripts in thermophilic anaerobic digester sludge. Different ammonium concentrations were checked for their influence on the methanogenic rate and hydrogen accumulation. Quantitative PCR was used to compare the changes in total archaeal 16S rRNA expression. A Monte Carlo permutation test within redundancy analysis (RDA) was adopted for exploring the relationship between environmental variables and archaeal 16S rRNA and their transcripts. The results showed that with the increase in ammonium concentration, the methanogenic rate decreased and hydrogen accumulation occurred. The total archaeal 16S rRNA genes and transcripts copy numbers decreased significantly in treatments with higher ammonium concentrations (7 and 10 g NH₄⁺-N/L), but did not change much at lower ammonia concentrations (3 g NH₄⁺-N/L) compared with the 0 g NH₄⁺-N/L treatment. The RDA analysis further revealed that most environmental variables, including ammonia and methane, except for formate, were significantly correlated with the community structure activity of archaeal 16S rRNA transcripts rather than the community structure of their genes. The composition of archaeal 16S rRNA transcripts showed that the hydrogenotrophic methanogen Methanothermobacter dominated the methanogenic community activity in all incubations. It exhibited sensitivity to ammonia stress and should be responsible for the methanogenic inhibition under thermophilic conditions. Our findings suggested that archaeal 16S rRNA transcripts, rather than 16S rRNA genes, are key indicators of ammonia stress and methanogenic activity.