Occurrence, Abundance, and Diversity of Tetracycline Resistance Genes in 15 Sewage Treatment Plants across China and Other Global Locations

Activated sludge was sampled from 15 sewage treatment plants (STPs) across China and other global locations to investigate the occurrence, abundance and diversity of tetracycline resistance genes (tet) in the STPs. Occurrence and abundance of 14 tet genes were determined using polymerase chain reaction (PCR) and quantitative real time PCR. Six genes (tet(A), tet(C), tet(G), tet(M), tet(S), and tet(X)) were detected in all the STPs, while no sludge sample contained tet(Q). Total concentration of the 14 genes was significantly different among the STPs and average tet abundance of the STPs varied greatly among the tet types (p Tet(G) had the highest concentration in the STPs, followed by tet(C), tet(A) and tet(S). Phylogenetic diversity of the genes was investigated using DNA cloning. BLAST analysis showed that all of the 450 cloned sequences matched known tet genes, except for tet(G). The 56 tet(G) clones were grouped into 14 genotypes, among which type G24 had an identical sequence to tet(G) carried by Salmonella enterica or Acinetobacter baumannii, while the other sequences had low similarity to the known genes in GenBank. The results of this study might be useful to understand the diversity of these resistance genes in STPs

Additional file 1 of Machine learning-aided analyses of thousands of draft genomes reveal specific features of activated sludge processes

Additional file 1: Table S1. Information about the WWTPs and activate sludge samples analyzed in this study. Table S2. Accession numbers of the metagenomic datasets used in this study. Table S3. Abundance of AS MAGs assigned to each phylum. Table S4. Prediction report of the random forest model. Table S5. Importance values and descriptions of the top 20 COGs identified by random forest model to differentiate the AS and non-AS MAGs. Figure S1. Geographical locations of the WWTPs where activated sludge samples were collected by us and other researchers. Figure S2. Associations between MAG completeness and number of contigs (a), and associations between MAG completeness and number of contigs (b). Figure S3. Venn diagram showing the shared and unique MAGs of WWTP1, WWTP2, WWTP3 and WWTP4. Figure S4. Profile of protein sequences identity between different WWTPs. The protein sequences predicted from all assembly contigs of each WWTP were compared each other with Diamond and then the best hits of the protein sequences were counted and summarized. Figure S5. Random forest parameter tuning and optimization. (a) Number of trees (n_estimators); (b) Tree depth; (c) Maximum features. Figure S6. Phylogeny of the erroneously predicted MAGs. The topology of this tree is exactly same with Fig. 1b. Extended lines were added to show positions of the erroneously predicted MAGs

Additional file 1 of Machine learning-aided analyses of thousands of draft genomes reveal specific features of activated sludge processes

Additional file 1: Table S1. Information about the WWTPs and activate sludge samples analyzed in this study. Table S2. Accession numbers of the metagenomic datasets used in this study. Table S3. Abundance of AS MAGs assigned to each phylum. Table S4. Prediction report of the random forest model. Table S5. Importance values and descriptions of the top 20 COGs identified by random forest model to differentiate the AS and non-AS MAGs. Figure S1. Geographical locations of the WWTPs where activated sludge samples were collected by us and other researchers. Figure S2. Associations between MAG completeness and number of contigs (a), and associations between MAG completeness and number of contigs (b). Figure S3. Venn diagram showing the shared and unique MAGs of WWTP1, WWTP2, WWTP3 and WWTP4. Figure S4. Profile of protein sequences identity between different WWTPs. The protein sequences predicted from all assembly contigs of each WWTP were compared each other with Diamond and then the best hits of the protein sequences were counted and summarized. Figure S5. Random forest parameter tuning and optimization. (a) Number of trees (n_estimators); (b) Tree depth; (c) Maximum features. Figure S6. Phylogeny of the erroneously predicted MAGs. The topology of this tree is exactly same with Fig. 1b. Extended lines were added to show positions of the erroneously predicted MAGs

Correlations of Gut Microbial Community Shift with Hepatic Damage and Growth Inhibition of <i>Carassius auratus</i> Induced by Pentachlorophenol Exposure

Goldfish (<i>Carassius auratus</i>) were exposed to 0–100 μg/L pentachlorophenol (PCP) for 28 days to investigate the correlations of fish gut microbial community shift with the induced toxicological effects. PCP exposure caused accumulation of PCP in the fish intestinal tract in a time- and dose-dependent manner, while hepatic PCP reached the maximal level after a 21 day exposure. Under the relatively higher PCP stress, the fish body weight and liver weight were reduced and hepatic CAT and SOD activities were inhibited, demonstrating negative correlations with the PCP levels in liver and gut content (<i>R</i> < −0.5 and <i>P</i> < 0.05 each). Pyrosequencing of the 16S rRNA gene indicated that PCP exposure increased the abundance of Bacteroidetes in the fish gut. Within the Bacteroidetes phylum, the <i>Bacteroides</i> genus had the highest abundance, which was significantly correlated with PCP exposure dosage and duration (<i>R</i> > 0.5 and <i>P</i> < 0.05 each). Bioinformatic analysis revealed that <i>Bacteroides</i> showed quantitatively negative correlations with <i>Chryseobacterium</i>, <i>Microbacterium</i>, <i>Arthrobacter</i>, and <i>Legionella</i> in the fish gut, and the Bacteroidetes abundance, <i>Bacteroides</i> abundance, and Firmicutes/Bacteroidetes ratio played crucial roles in the reduction of body weight and liver weight under PCP stress. The results may extend our knowledge regarding the roles of gut microbiota in ecotoxicology

Bacterial Community Shift Drives Antibiotic Resistance Promotion during Drinking Water Chlorination

For comprehensive insights into the effects of chlorination, a widely used disinfection technology, on bacterial community and antibiotic resistome in drinking water, this study applied high-throughput sequencing and metagenomic approaches to investigate the changing patterns of antibiotic resistance genes (ARGs) and bacterial community in a drinking water treatment and distribution system. At genus level, chlorination could effectively remove <i>Methylophilus</i>, <i>Methylotenera</i>, <i>Limnobacter</i>, and <i>Polynucleobacter</i>, while increase the relative abundance of <i>Pseudomonas</i>, <i>Acidovorax</i>, <i>Sphingomonas</i>, <i>Pleomonas</i>, and <i>Undibacterium</i> in the drinking water. A total of 151 ARGs within 15 types were detectable in the drinking water, and chlorination evidently increased their total relative abundance while reduced their diversity in the opportunistic bacteria (<i>p</i> < 0.05). Residual chlorine was identified as the key contributing factor driving the bacterial community shift and resistome alteration. As the dominant persistent ARGs in the treatment and distribution system, multidrug resistance genes (mainly encoding resistance-nodulation-cell division transportation system) and bacitracin resistance gene <i>bac</i>A were mainly carried by chlorine-resistant bacteria <i>Pseudomonas</i> and <i>Acidovorax</i>, which mainly contributed to the ARGs abundance increase. The strong correlation between bacterial community shift and antibiotic resistome alteration observed in this study may shed new light on the mechanism behind the chlorination effects on antibiotic resistance

Inhibition of Mitochondrial Fatty Acid Oxidation Contributes to Development of Nonalcoholic Fatty Liver Disease Induced by Environmental Cadmium Exposure

Cadmium (Cd) is one of the most prevalent toxic metal pollutants widely distributed in water and soil environments. Epidemiological studies have shown that exposure to Cd is implicated in the prevalence of nonalcoholic fatty liver disease (NAFLD) in middle-aged human population, but biological evidence is lacking and its toxicological mechanism remains unclear for the disease predisposition from environmental Cd exposure. In this study, we established a chronic Cd-exposure mouse model mimicking the liver Cd deposition in middle-aged human population to determine whether the environmental Cd exposure can induce NAFLD. Results showed that hepatic Cd burden at levels of 0.95 and 6.04 μg/g wet weight resulting from 20-week Cd exposure at different doses induced NAFLD and nonalcoholic steatohepatitis-like phenotypes in mice, respectively. The Cd exposure caused marked hepatic mitochondrial dysfunction and fatty acid oxidation deficiency, along with significant suppression of sirtuin 1 (SIRT1) signaling pathway in the liver. In vitro study confirmed that Cd evidently inhibited the mitochondrial fatty acid oxidation in hepatocytes and that SIRT1 signaling was potentially involved in the process. Our findings suggest that exposure to environmental Cd is a tangible risk factor for NAFLD, and the induced public health risks deserve greater attention

Microcystin-LR Promotes Melanoma Cell Invasion and Enhances Matrix Metalloproteinase-2/‑9 Expression Mediated by NF-κB Activation

This study aimed to explore the molecular mechanisms behind the stimulation effects of microcystin-LR (a well-known cyanobacterial toxin produced in eutrophic lakes or reservoirs) on cancer cell invasion and matrix metalloproteinases (MMPs) expression. Boyden chamber assay showed that microcystin-LR exposure (>12.5 nM) evidently enhanced the invasion ability of the melanoma cells (MDA-MB-435). Tumor Metastasis PCR Array demonstrated that 24 h microcystin-LR treatment (25 nM) caused overexpression of eight genes involved in tumor metastasis, including MMP-2, MMP-9, and MMP-13. Quantitative real-time PCR, Western blotting and gelatin zymography consistently demonstrated that mRNA and protein levels of MMP-2/-9 were increased in the cells after microcystin-LR exposure (<i>P</i> < 0.05 each). Immunofluorescence assay and electrophoretic mobility shift assay revealed that microcystin-LR could activate nuclear factor kappaB (NF-κB) by accelerating NF-κB translocation into the nucleus and enhancing NF-κB binding ability. Furthermore, addition of NF-κB inhibitor in culture medium could suppress the invasiveness enhancement and MMP-2/-9 overexpression. This study indicates that microcystin-LR can act as a NF-κB activator to promote MMP-2/-9 expression and melanoma cell invasion, which deserves more environmental health concerns

Abundance and Diversity of Bacterial Nitrifiers and Denitrifiers and Their Functional Genes in Tannery Wastewater Treatment Plants Revealed by High-Throughput Sequencing

<div><p>Biological nitrification/denitrification is frequently used to remove nitrogen from tannery wastewater containing high concentrations of ammonia. However, information is limited about the bacterial nitrifiers and denitrifiers and their functional genes in tannery wastewater treatment plants (WWTPs) due to the low-throughput of the previously used methods. In this study, 454 pyrosequencing and Illumina high-throughput sequencing, combined with molecular methods, were used to comprehensively characterize structures and functions of nitrification and denitrification bacterial communities in aerobic and anaerobic sludge of two full-scale tannery WWTPs. Pyrosequencing of 16S rRNA genes showed that <i>Proteobacteria</i> and <i>Synergistetes</i> dominated in the aerobic and anaerobic sludge, respectively. Ammonia-oxidizing bacteria (AOB) <i>amoA</i> gene cloning revealed that <i>Nitrosomonas europaea</i> dominated the ammonia-oxidizing community in the WWTPs. Metagenomic analysis showed that the denitrifiers mainly included the genera of <i>Thauera</i>, <i>Paracoccus</i>, <i>Hyphomicrobium</i>, <i>Comamonas</i> and <i>Azoarcus</i>, which may greatly contribute to the nitrogen removal in the two WWTPs. It is interesting that AOB and ammonia-oxidizing archaea had low abundance although both WWTPs demonstrated high ammonium removal efficiency. Good correlation between the qPCR and metagenomic analysis is observed for the quantification of functional genes <i>amoA</i>, <i>nirK</i>, <i>nirS</i> and <i>nosZ</i>, indicating that the metagenomic approach may be a promising method used to comprehensively investigate the abundance of functional genes of nitrifiers and denitrifiers in the environment.</p></div

Neighbor-joining phylogenetic tree (A) and OTUs number (B) of AOB <i>amoA</i> gene in the four sludge samples.

<p>The evolutionary distances were computed using the Jukes–Cantor method. Bootstrap values (over 50) are indicated on branch nodes. Sequences obtained from this study are shown with “<i>OTU-</i>” in the names, and reference sequences were obtained from GenBank.</p

Heat map illustrating the abundance of all the major genera (with relative abundance over 1% in at least one sample).

<p>The color intensity (log scale) in each panel indicates the relative abundance of the genus in each sample.</p