Characterization of a novel tetravalent botulism antitoxin based on receptor-binding domain of BoNTs.

Botulinum neurotoxin (BoNTs; serotypes A, B, E, and F) cause botulism disease in humans, which could be effectively treated using antitoxins. Herein, we established a novel receptor-binding domain (RBD)-based antitoxin using recombinant C terminal heavy chain (Hc) domains of BoNTs as immunogens. Immunization of horses with these recombinant Hc domains allowed the purification and digestion of IgGs from hyper-immune sera to produce high-quality and high-efficiency monovalent botulism antitoxin F(ab')2 against each BoNT (M-BATs). However, these M-BATs could not bind or neutralize other serotypes of BoNTs, and that there were no cross-protective effects among these M-BATs. This suggested the need to prepare tetravalent antitoxins to neutralize the four BoNTs simultaneously. Thus, these M-BATs were formulated into a novel tetravalent botulism antitoxin (T-BAT), in which a 10-ml volume contained 10000 IU of BoNT/A and 5000 IU of BoNT/B, BoNT/E, and BoNT/F antitoxins. The novel antitoxin preparation could prevent and treat the four mixed botulinum neurotoxins simultaneously in vivo, representing strong efficacy in an animal poisoning model. Moreover, these antibodies in T-BAT could bind the RBD, whereas conventional antitoxins based on inactivated toxins mainly bind the light chain or heavy chain translocation domain (HN) and weakly bind the important RBD in current experimental conditions. The high levels of RBD-specific novel antitoxins can efficiently bind the RBD and neutralize natural or recombinant toxins containing this RBD. The findings of the present study experimentally support the use of RBD-specific antitoxins to treat BoNT serotype A, B, E, and F-mediated botulism. This study demonstrated the concept of developing potent novel multivalent antitoxins against all BoNTs or other toxins, using the RBD of these toxins as an alternative antigen to inactivated toxins. KEY POINTS: • Antitoxins based on the receptor-binding domains of botulinum neurotoxins were made. • Novel antitoxin binds RBD; traditional antitoxin mainly binds light chain or HN domain. • A tetravalent antitoxin could prevent and treat the four mixed neurotoxins in vivo.

Antibiotic resistance genes in gut of breast-fed neonates born by caesarean section originate from breast milk and hospital ward air.

The human gut is a reservoir of antibiotic resistance genes (ARGs). Even in the absence of antibiotics, ARGs are present in large quantities in faeces of adults, children and even newborns. However, where and when ARGs are acquired remains unclear, as does the types of ARGs acquired. Herein, we recruited 82 pairs of women and their caesarean section newborns. Conventional culture methods and quantitative PCR were employed to detect nine species and six ARG types in meconia, faeces from 3-day-old newborns, amniotic fluid, colostrum, and hospital ward air samples. Furthermore, ARG transfer was explored by tracking Staphylococcus epidermidis isolated from faeces of 3-day-old newborns, colostrum and ward air samples using multi-locus sequence typing (MLST). No ARGs or microorganisms were detected in meconia or amniotic fluid. One or more ARGs were detected in 90.2% of faeces from 3-day-old newborns, and the mecA gene exhibited the highest detection rate (45.1%). ARGs were detected in 85.4% of colostra consistent with ARGs in faeces from 3-day-old newborns. Some ARGs were detected in ward air, and might also be a source of ARGs in neonatal faeces. Isolation of S. epidermidis from neonatal faeces was consistent with antibiotic resistance and gene profiles for colostrum samples. Traceability analysis of S. epidermidis showed that ARGs in neonatal faeces mainly originated from colostrum, and partly from ward air. After birth, neonates born by caesarean section obtain a variety of ARGs mainly from colostrum, and partly from ward air.

Human Papillomavirus E6/E7 and Long Noncoding RNA TMPOP2 Mutually Upregulated Gene Expression in Cervical Cancer Cells.

TMPOP2 was previously suggested to be an oncogenic long noncoding RNA which is excessively expressed in cervical cancer cells and inhibits E-cadherin gene expression by recruiting transcription repressor EZH2 to the gene promoter. So far, the function and regulation of TMPOP2 in cervical cancer remain largely unknown. Herein, we found that TMPOP2 expression was correlated with human papillomavirus 16/18 (HPV16/18) E6 and E7 in cervical cancer cell lines CaSki and HeLa. Tumor suppressor p53, which is targeted for degradation by HPV16/18, was demonstrated to associate with two p53 response elements in the TMPOP2 promoter to repress the transcription of the TMPOP2 gene. Reciprocally, ectopic expression of TMPOP2 was demonstrated to sequester tumor repressor microRNAs (miRNAs) miR-375 and miR-139 which target HPV16/18 E6/E7 mRNA and resulted in an upregulation of HPV16/18 E6/E7 genes. Thereby, HPV16/18 E6/E7 and the long noncoding RNA (lncRNA) TMPOP2 form a positive feedback loop to mutually derepress gene expression in cervical cancer cells. Moreover, results of RNA sequencing and cell cycle analysis showed that knockdown of TMPOP2 impaired the expression of cell cycle genes, induced cell cycle arrest, and inhibited HeLa cell proliferation. Together, our results indicate that TMPOP2 and HPV16/18 E6/E7 mutually strengthen their expression in cervical cancer cells to enhance tumorigenic activities.IMPORTANCE Human papillomaviruses 16 and 18 (HPV16/18) are the main causative agents of cervical cancer. Viral proteins HPV16/18 E6 and E7 are constitutively expressed in cancer cells to maintain oncogenic phenotypes. Accumulating evidences suggest that HPVs are correlated with the deregulation of long noncoding RNAs (lncRNAs) in cervical cancer, although the mechanism was unexplored in most cases. TMPOP2 is a newly identified lncRNA excessively expressed in cervical cancer. However, the mechanism for the upregulation of TMPOP2 in cervical cancer cells remains largely unknown and its relationship with HPVs is still elusive. The significance of our research is in revealing the mutual upregulation of HPV16/18 E6/E7 and TMPOP2 with the molecular mechanisms explored. This study will expand our understandings of the oncogenic activities of human papillomaviruses and lncRNAs.

Effects of free antibiotic resistance genes in the environment on intestinal microecology of mice.

The rapid spread of antibiotic resistance genes (ARGs) is a great challenge to the ecological safety and human health. The intestine of humans and animals is an important site for the increase and spread of ARGs due to the great diversity and abundance of microorganisms in the intestinal microecology. ARGs, including the intracellular (iARGs) and the extracellular (eARGs) ARGs, are usually introduced into the intestinal tract through the diet, and the iARGs are colonized and spread in the intestinal microbiota with the help of the host bacteria. However, whether the eARGs can enter the intestinal microorganisms in the absence of host bacteria is not known. Here, we show the transformation and the diffusion of the ampramycin resistance gene (Ap) carried by the free plasmid RK2 in the intestinal microbiota of mice. After two days of consecutive gavage with free RK2, the intracellular Ap gene increases from days 0-8 in the feces of mice, and has remained constant. Bacterial transformation happens in the small intestine, including proximal and distal jejuna and proximal and distal ilea, at the early stage (first two days), and the intracellular RK2 is diffused into the intestinal microbiota of mice by conjugation on days 2-8 day, which is based on the distribution of eARG and iARG and the mRNA expression levels of trbBp, trfAp, korA, korB, and trbA. The characteristics of ARGs susceptible microbiota for transformation are analyzed using 16s rRNA gene sequencing, transmission electron microscopy, and flow cytometric. The ingestion of RK2 affects the composition of intestinal microbiota especially for Proteobacteria, and the antibiotic residue promotes the increase in Escherichia coli. These findings are important to assess the risk of ARGs, especially the eARGs in the intestinal microecology.

Putative environmental levels of levofloxacin facilitate the dissemination of antibiotic-resistant Escherichia coli via plasmid-mediated transformability.

Antibiotic residues in the environment pose a great risk to global public health. They increase antibiotic resistance by enhancing plasmid conjugation among bacteria or mutations within bacterial genomes. However, little is known about whether the putative environmental levels of antibiotics are sufficient to influence plasmid-mediated transformability. In this study, we explored the effect of eight kinds of representative antibiotics and several other compounds on the plasmid transformability of competent Escherichia coli. Only levofloxacin (LEV) at the putative environmental levels was found to facilitate the frequency of PBR322-or RP4-plasmid-mediated transformation by up to 5.3-fold. Additionally, PBR322 transformation frequency could be further enhanced by copper ion or ammonia nitrogen but inhibited by humic acid. However, when competent E. coli was exposed to the minimal inhibitory concentrations (MIC) of the antibiotics, an enhanced plasmid-assimilation ability was observed and plasmid transformation frequency was increased by up to 98.6-fold for all the tested antibiotics. Furthermore, E. coli exhibited a preference for the uptake of plasmids harbouring the resistance genes to the antibiotics it had been exposed to. Among these antibiotics, cephalexin, tetracycline, and kanamycin induced the highest uptake of RP4. The putative environmental levels of LEV enhanced plasmid transformability regardless of the presence of corresponding antibiotic resistance gene (ARG) on the genetic elements, suggesting environmental LEV residues may facilitate dissemination of antibiotic resistance by any plasmid-mediated transformability, thereby posing a great risk to health.

Silymarin attenuates paraquat-induced lung injury via Nrf2-mediated pathway in vivo and in vitro.

The present study aims to investigate the impacts and mechanisms of silymarin on paraquat (PQ)-induced lung injury in vivo and in vitro. In in vivo experiments, a total of 32 male Sprague-Dawley (SD) rats were randomly divided into four groups. The rats were killed on day 3. Histopathological changes in lung tissue were examined using HE and Masson's trichrome staining. Biomarkers of neutrophil activation, pulmonary oedema, pulmonary fibrosis, lung permeability and oxidative stress were detected. Several proinflammatory mediators and antioxidant related proteins were measured. In in vitro experiments, A549 cells were transfected with Nrf2 special siRNA to investigate the roles of Nrf2. The results show that silymarin administration abated PQ-induced lung histopathologic changes, decreased inflammatory cell infiltration and lung wet weight/dry weight (W/D) ratio, suppressed myeloperoxidase (MPO) activity and nitric oxide (NO)/inducible nitric oxide synthases (iNOS) expression, downregulated hydroxyproline (HYP) levels, reduced total protein concentration and proinflammatory mediator release, and improved oxidative stress (malondialdehyde, MDA; superoxide dismutase, SOD; catalase, CAT; and glutathione peroxidase, GSH-Px) in lung tissue and serum. Meanwhile, treatment with silymarin upregulated the levels of nuclear factor-erythroid-2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1(NQO1). However, the addition of Nrf2 siRNA reduced the expression of Nrf2-mediated antioxidant protein HO-1 and thus reversed the protective effects of silymarin against oxidative stress and inflammatory response. These results suggest that silymarin may exert protective effects against PQ-induced lung injury. Its mechanisms were associated with the Nrf2-mediated pathway. Therefore, silymarin may be a potential therapeutic drug for lung injury.

CRISPR/Cas9-based engineered Escherichia coli biosensor for sensitive and specific detection of Cd(II) in drinking water.

Cadmium (Cd) is a ubiquitous pollutant that poses a potential threat to human health. Monitoring Cd(II) in drinking water has significant implications for preventing potential threats of Cd(II) to human. However, the weak signal output and response to nontarget interference limit the detection of Cd(II) using bacterial biosensors. In this study, to enable sensitive and specific detection of Cd(II) in water, a stable whole-cell biosensor, K12-PMP-luxCDABE-△cysI, was constructed in a dual-promoter mode by fusing the mercury promoter Pmer, regulatory gene merR(m), and luciferase gene luxCDABE into the E.coli chromosome based on CRISPR/Cas9 gene editing technology. By knocking out the cadmium-resistance-gene cysI, the sensitivity of the biosensor to Cd(II) was further enhanced. The constructed E. coli biosensor K12-PMP-luxCDABE-△cysI exhibited good nonlinear responses to 0.005-2 mg/L Cd(II). Notably, among the three constructed E. coli biosensor, it exhibited the strongest fluorescence intensity, with the limit of detection meeting the allowable limit for Cd(II) in drinking water. Simultaneously, it could specifically detect Cd(II). Nontarget metal ions, such as Zn(II), Hg(II), and Pb(II), did not affect its performance. Furthermore, it exhibited superior performance in detecting Cd(II) in real drinking water samples by avoiding background interference, and showed excellent stability with the relative standard deviation under 5%. Thus, K12-PMP-luxCDABE-△cysI holds promise as a potential tool for the detection of Cd(II) in drinking water.

Metagenomics of high-altitude groundwater reveal different health risks associated with antibiotic-resistant pathogens and bacterial resistome in the latitudinal gradient.

Groundwater on the Tibetan Plateau is a critical water resource to people in Asia. However, its prevalence of antibiotic-resistant pathogens (ARPs), bacterial resistome and their driving factors remain unknown. Using metagenomics analysis, a hotspot of antibiotic-resistance genes (ARGs) and last-resort ARGs (LARGs) with a total of 639 subtypes was identified in the groundwater. Importantly, 164 metagenome-assembled genomes (MAGs) which possessed both ARGs and virulence factors (VFs) were assigned as potential ARPs, with the most abundant species being Acinetobacter johnsonii and Acinetobacter pittii. A total of 157 potential ARPs, involving Escherichia coli, were predicted as "natural" ARGs supercarriers. Thirty-six ARPs dominated by the genus Acinetobacter and Pseudomonas were found to harbour LARGs. Co-localizations of the ARG-mobile genetic elements (MGEs) showed that MGEs were significantly associated with ARGs in the ARPs, which suggests ARPs play a prominent role in ARG dissemination. Notably, latitudinal gradient is a driving factor in the occurrence of ARGs and ARPs. The average abundances of ARGs and ARP decreased as the latitude increased, with the highest abundance occurring in the region between 28.6◦N and 29.5◦N. MetaCompare further revealed health risks associated with the resistome decreased as the latitudes increased. These findings indicated different health risks associated with ARPs and bacterial resistome in latitudinal gradient groundwater. They raise the concerns of mitigating ARPs risk in groundwater on the Tibetan Plateau.

A mica filter enables bacterial enrichment from large volumes of natural water for sensitive monitoring of pathogens by nanopore sequencing.

Nanopore sequencing is extremely promising for the high-throughput detection of pathogenic bacteria in natural water; these bacteria may be transmitted to humans and cause waterborne infectious diseases. However, the concentration of pathogenic bacteria in natural water is too low to be detected directly by nanopore sequencing. Herein, we developed a mica filter to enrich over 85% of bacteria from > 10 L of natural water in 100 min, which led to a 102-fold improvement in the assay limits of the MinION sequencer for assessing pathogenic bacteria. Correspondingly, the sequencing time of S. Typhi detection at a concentration as low as 105 CFU/L was reduced from traditional 48 h to 3 h. The bacterial adsorption followed pseudo-first-order kinetics and the successful adsorption of bacteria to the mica filter was confirmed by scanning electron microscopy and Fourier infrared spectroscopy et al. The mica filter remained applicable to a range of water samples whose quality parameters were within the EPA standard limits for freshwater water. The mica filter is thus an effective tool for the sensitive and rapid monitoring of pathogenic bacteria by nanopore sequencing, which can provide timely alerts for waterborne transmission events.

Emergence of colistin-resistant Stenotrophomonas maltophilia with high virulence in natural aquatic environments.

The presence of Stenotrophomonas maltophilia in aquatic environments poses great health risks to immunocompromised individuals because of its multidrug resistance and resultant high mortality. However, a significant gap exists in the isolation and understanding of colistin-resistant S. maltophilia in aquatic environments. In this study, nine colistin-resistant S. maltophilia strains isolated from natural lakes were explored, and their phylogenetic relationship, biofilm formation, virulence, and antibiotic resistance profiles and underlying genetic determinants were assessed. After genome analysis, besides known multi-locus sequence typing (MLST) of ST532, new assigned ST965 and ST966 which phylogenetically clustered into soil isolates were found firstly. All the isolates exhibited resistance to multiple antibiotics, including aminoglycosides, beta-lactams, tetracyclines, and even colistin, with the highest minimum inhibitory concentration (MIC) against colistin reaching 640 mg/L. Comparative genomic analysis revealed aph(3')-Iic, blaL1, tetT, phoP, mcr-3, arnA, pmrE, and efflux pump genes as the genetic determinants underlying this multidrug resistance. Notably, the biofilm-forming capacities of the newly discovered ST965 and ST966 isolates were significant stronger than those of the known ST532 isolates (p < 0.01), resulting in the death of over 50 % of the Galleria mellonella population within 1 day of injection. The ST965 isolates demonstrated the highest virulence against G. mellonella, followed by the ST966 isolates and ST532 isolates which was phylogenetically clustered with clinical isolates, indicating that the novel S. maltophilia strains of ST965 and ST966 may pose considerable health risks to humans. Our findings provide insights into colistin-resistant S. maltophilia in aquatic environments and raise concerns about the health risks posed by the newly assigned sequence types of colistin-resistant S. maltophilia with potential high virulence in natural aquatic environments.

Carbonaceous particulate matter promotes the horizontal transfer of antibiotic resistance genes.

There is growing concern about the transfer of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in airborne particulate matter. In this study, we investigated the effects of various types of carbonaceous particulate matter (CPM) on the transfer of ARGs in vitro. The results showed that CPM promoted the transfer of ARGs, which was related to the concentration and particle size. Compared with the control group, the transfer frequency was 95.5, 74.7, 65.4, 14.7, and 3.8 times higher in G (graphene), CB (carbon black), NGP (nanographite powder), GP1.6 (graphite powder 1.6 micron), and GP45 (graphite powder 45 micron) groups, respectively. Moreover, the transfer frequency gradually increased with the increase in CPM concentration, while there was a negative relationship between the CPM particle size and conjugative transfer frequency. In addition, the results showed that CPM could promote the transfer of ARGs by increasing ROS, as well as activating the SOS response and expression of conjugative transfer-related genes (trbBp, trfAp, korA, kroB, and trbA). These findings are indicative of the potential risk of CPM for the transfer of ARGs in the environment, enriching our understanding of environmental pollution and further raising awareness of environmental protection.

Comparative analysis of chlorine-resistant bacteria after chlorination and chloramination in drinking water treatment plants.

Chlorine-resistant bacteria (CRB) in drinking water treatment plants (DWTPs) jeopardize water quality and pose a potential risk to human health. However, the specific response of CRB to chlorination and chloramination remains uncharacterized. Therefore, we analyzed 16 S rRNA sequencing data from water samples before and after chlorination and chloramination taken between January and December 2020. Proteobacteria and Firmicutes dominated all finished water samples. After chloramination, Acinetobacter, Pseudomonas, Methylobacterium, Ralstonia, and Sphingomonas were the dominant CRB, whereas Ralstonia, Bacillus, Acinetobacter, Pseudomonas, and Enterococcus were prevalent after chlorination. Over 75% of the CRB e.g. Acinetobacter, Pseudomonas, Bacillus, and Enterococcus were shared between the chlorination and chloramination, involving potentially pathogens, such as Acinetobacter baumannii and Pseudomonas aeruginosa. Notably, certain genera such as Faecalibacterium, Geobacter, and Megasphaera were enriched as strong CRB after chloramination, whereas Vogesella, Flavobacterium, Thalassolituus, Pseudoalteromonas, and others were enriched after chlorination according to LEfSe analysis. The shared CRB correlated with temperature, pH, and turbidity, displaying a seasonal pattern with varying sensitivity to chlorination and chloramination in cold and warm seasons. These findings enhance our knowledge of the drinking water microbiome and microbial health risks, thus enabling better infectious disease control through enhanced disinfection strategies in DWTPs.

Determination of total phenol and six polyphenolic components in the polyphenol extract of Cinnamomi cortex by quantitative nuclear magnetic resonance spectroscopy.

A quantitative nuclear magnetic resonance spectroscopy (qNMR) method was established for determining the total phenol and six polyphenolic components in the polyphenol extract of Cinnamomi cortex. The qNMR approach utilized DMSO-d6 as the deuterated solvent and potassium hydrogen phthalate as the internal standard for quantifying the total phenolic content, expressed as epicatechin equivalence in the sample. Two complementary qNMR methods with DMSO-d6 or D2O as solvent were established to simultaneously determine 6 polyphenol components in the cinnamon polyphenol extract, including epigallocatechin gallate (EGCG), epicatechingallate (ECG), epicatechin (EC), epigallocatechin (EGC), gallocatechin gallate (GCG) and gallic acid (GA). Method validation demonstrated excellent precision with intraday relative standard deviation (RSD) below 1.08% and interday RSD below 1.48%. The linear correlation coefficient (r) exceeded 0.999, and the limits of detection (LOD) were from 0.01 to 0.14 mg mL-1, while the limits of quantification (LOQ) were from 0.07 to 0.69 mg mL-1. Recovery rates for this method fell within the range of 98.2% to 101.7%. Furthermore, the method has been successfully applied for determining the polyphenolic content in authentic cinnamon polyphenol extracts obtained from different sources.

Representation Learning on Heterostructures via Heterogeneous Anonymous Walks.

Capturing structural similarity has been a hot topic in the field of network embedding (NE) recently due to its great help in understanding node functions and behaviors. However, existing works have paid very much attention to learning structures on homogeneous networks, while the related study on heterogeneous networks is still void. In this article, we try to take the first step for representation learning on heterostructures, which is very challenging due to their highly diverse combinations of node types and underlying structures. To effectively distinguish diverse heterostructures, we first propose a theoretically guaranteed technique called heterogeneous anonymous walk (HAW) and give two more applicable variants. Then, we devise the HAW embedding (HAWE) and its variants in a data-driven manner to circumvent using an extremely large number of possible walks and train embeddings by predicting occurring walks in the neighborhood of each node. Finally, we design and apply extensive and illustrative experiments on synthetic and real-world networks to build a benchmark on heterostructure learning and evaluate the effectiveness of our methods. The results demonstrate our methods achieve outstanding performance compared with both homogeneous and heterogeneous classic methods and can be applied on large-scale networks.

Landscape of antibiotic resistance genes and bacterial communities in groundwater on the Tibetan Plateau, and distinguishing their difference with low-altitude counterparts.

Groundwater is a vital source of drinking water for Tibetans. Antibiotic resistance genes (ARGs) and bacterial communities in groundwater on the Tibetan Plateau remain unclear. Furthermore, the characterization of their differences between high-altitude and low-altitude groundwater is still unrevealed. Herein, 32 groundwater samples were collected on the plateau, and intra- and extracellular ARGs (iARGs and eARGs), and bacterial communities were characterised through qPCR assays to 19 ARGs and 16S rRNA sequencing. It showed top four abundant intra- and extracellular last-resort ARGs (LARGs) were blaOXA-48, mcr-1, vanA, and vanB, whereas dominant common ARGs (CARGs) were tetA and ermB, respectively. CARGs had higher abundances than LARGs, and iARGs were more frequently detected than eARGs. Proteobacteria, an invasive resident phylum, and Firmicutes dominated eDNA release. Network analysis revealed all observed LARGs co-occurred with pathogenic and non-pathogenic bacteria. Community diversity was significantly associated with longitude and elevation, while nitrate correlated with ARGs. Comparative analysis demonstrated eARG frequencies and abundances were higher at high altitudes than at low altitudes. Additionally, Acinetobacter and Pseudomonas specifically dominated at high altitudes. This study reveals the widespread prevalence of ARGs, particularly LARGs, in groundwater on the less-disturbed Tibetan Plateau and underlines the potential risks associated with the LARG-carrying bacteria. ENVIRONMENTAL IMPLICATION: Antibiotic resistance genes (ARGs), which are defined as emerging environmental contaminants, are becoming a global concern due to their ability to confer antibiotic resistance to pathogens. Our findings highlight the prevalence of ARGs, particularly LARGs, in groundwater on the Tibetan Plateau, and the possibility that naturally-occurring pathogenic and non-pathogenic bacteria carry multiple LARGs. In addition, we further reveal differences in the distribution of ARGs and bacterial community between high-altitude and low-altitude groundwater. Collectively, our findings offer an important insight into the potential public risks related to groundwater on the Tibetan Plateau.

Spatial and temporal analysis of the seasonal dynamics of antibiotic resistance gene occurrence in recreational marine water.

People who engage in water sports in recreational marine water may be at high risk of exposure to hazardous antibiotic-resistant bacteria (ARB). However, information on the contribution of specific sources to ARB contamination in recreational marine water is still lacking. Here, we carried out monthly analyses of antibiotic resistance genes (ARGs), pathogenic bacteria and 16S rRNA sequencing data at the First Bathing Beach in Qingdao. The sampling sites were divided into four areas: swimming area, intermediate area, polluted area, and sewage outlet. Correlations between ARGs and bacterial communities among sampling sites were explored by spatial and temporal analysis. We found that all of 21 important ARG types were detected in the swimming area, with aadA (1.3 × 106 ± 2.7 × 106 genomic copies/L) and sul2 (4.3 × 105 ± 5.9 × 105 genomic copies/L) at the highest concentration. Most ARGs were detected at highest frequency and concentration in the sewage outlet and decreased from there to the swimming area. ARG correlation between these two areas was positive only in the cold season, suggesting that sewage was the main source of ARG pollution in the swimming area during that period. The ARGs ermA(1) and vanA were detected at highest frequency and concentration in the swimming area and were significantly correlated with the intestinal pathogen Enterococcus, which was more abundant here than in the surrounding areas during the warm season. Co-occurrence analysis of bacterial genera and ARGs showed that six genera were commonly correlated with ARGs in all sampling areas in the cold season, while none were found in the warm season. Our findings indicate that ARG pollution in the swimming area was also driven by sources other than sewage, especially in the warm season, which is the peak tourist season in Qingdao. These results provide a valuable basis for the implementation of effective strategies to control ARG risks in recreational waters.

Antidepressant exposure as a source of disinfectant resistance in waterborne bacteria.

The emergence of disinfectant-resistant pathogens in water is a major threat to public health. However, whether human-consumed pharmaceuticals can induce bacterial resistance to disinfectants remains unclear. Herein, Escherichia coli was exposed to 12 antidepressants, and susceptibility of antidepressant-induced chloramphenicol (CHL)-resistant mutants to disinfectants was tested. Whole genome sequencing, global transcriptomic sequencing, and real-time quantitative polymerase chain reaction were used to elucidate the underlying mechanisms. We observed that duloxetine, fluoxetine, amitriptyline, and sertraline significantly increased the mutation frequency of E. coli against CHL by 15- to 2948-fold. The resultant mutants increased the average MIC50 of sodium hypochlorite, benzalkonium bromide, and triclosan roughly 2- to 8-fold. Consistently, marRAB and acrAB-tolC genes, together with ABC transporter genes (e.g., yddA, yadG, yojI, and mdlA), were triggered to increase the efflux of disinfectants out of the cell, while ompF was inhibited, reducing disinfectant penetration into the cell. Additionally, the occurrence of DNA mutations in marR and acrR in the mutants was observed, potentially resulting in increased synthesis of the AcrAB-TolC pump. This study indicates that pharmaceutical exposure may create disinfectant-resistant bacteria, which may then be released into water systems, providing novel insights into the potential source of water-borne disinfectant-resistant pathogens.

Extended chloramination significantly enriched intracellular antibiotic resistance genes in drinking water treatment plants.

Chloramination and chlorination are both strong barriers that prevent the transmission of potential pathogens to humans through drinking water. However, the comparative effects of chloramination and chlorination on the occurrence of antibiotic resistance genes (ARGs) in drinking water treatment plants (DWTPs) remain unknown. Herein, the antibiotic resistome in water before and after chloramination or chlorination was analyzed through metagenomic sequencing and then verified through quantitative real-time polymerase chain reaction (qPCR). After the treatment of 90 min, chloramination led to higher enrichment of the total relative abundance of intracellular ARGs (iARGs) in water than chlorination, whereas chlorination facilitated the release of more extracellular ARGs (eARGs) than chloramination. According to redundancy and Pearson's analyses, the total concentration of the observed iARGs in the finished water exhibited a strong positive correlation with ammonium nitrogen (NH4+-N) concentration, presenting a linear upward trend with an increase in the NH4+-N concentration. This indicated that NH4+-N is a crucial driving factor for iARG accumulation during chloramination. iARG enrichment ceases if the duration of chloramination is shortened to 40 min, suggesting that shortening the duration would be a better strategy for controlling iARG enrichment in drinking water. These findings emphasized the potential risk of antibiotic resistance after extended chloramination, shedding light on the control of transmission of antibiotic-resistant bacteria through water by optimizing disinfection procedures in DWTPs.

Simulated Gastric Acid Promotes the Horizontal Transfer of Multidrug Resistance Genes across Bacteria in the Gastrointestinal Tract at Elevated pH Levels.

The assessment of factors that can promote the transmission of antibiotic resistance genes (ARGs) across bacteria in the gastrointestinal tract is in great demand to understand the occurrence of infections related to antibiotic-resistant bacteria (ARB) in humans. However, whether acid-resistant enteric bacteria can promote ARG transmission in gastric fluid under high-pH conditions remains unknown. This study assessed the effects of simulated gastric fluid (SGF) at different pH levels on the RP4 plasmid-mediated conjugative transfer of ARGs. Moreover, transcriptomic analysis, measurement of reactive oxygen species (ROS) levels, assessment of cell membrane permeability, and real-time quantitative assessment of the expression of key genes were performed to identify the underlying mechanisms. The frequency of conjugative transfer was the highest in SGF at pH 4.5. Antidepressant consumption and certain dietary factors further negatively impacted this situation, with 5.66-fold and 4.26-fold increases in the conjugative transfer frequency being noted upon the addition of sertraline and 10% glucose, respectively, compared with that in the control group without any additives. The induction of ROS generation, the activation of cellular antioxidant systems, increases in cell membrane permeability, and the promotion of adhesive pilus formation were factors potentially contributing to the increased transfer frequency. These findings indicate that conjugative transfer could be enhanced under certain circumstances in SGF at elevated pH levels, thereby facilitating ARG transmission in the gastrointestinal tract. IMPORTANCE The low pH of gastric acid kills unwanted microorganisms, in turn affecting their inhabitation in the intestine. Hence, studies on the factors that influence antibiotic resistance gene (ARG) propagation in the gastrointestinal tract and on the underlying mechanisms are limited. In this study, we constructed a conjugative transfer model in the presence of simulated gastric fluid (SGF) and found that SGF could promote the dissemination of ARGs under high-pH conditions. Furthermore, antidepressant consumption and certain dietary factors could negatively impact this situation. Transcriptomic analysis and a reactive oxygen species assay revealed the overproduction of reactive oxygen species as a potential mechanism by which SGF could promote conjugative transfer. This finding can help provide a comprehensive understanding of the bloom of antibiotic-resistant bacteria in the body and create awareness regarding the risk of ARG transmission due to certain diseases or an improper diet and the subsequent decrease in gastric acid levels.

Emerging pollutant metformin in water promotes the development of multiple-antibiotic resistance in Escherichia coli via chromosome mutagenesis.

Antibiotics are known to be key drivers of antibiotic resistance and antibiotic resistance gene transmission. However, the contribution of the emerging pollutant metformin in facilitating antibiotic resistance remains unclear. In this study, Escherichia coli K12 (E. coli) was exposed to metformin at concentrations ranging from 10-7 to 200 mg/L, and antibiotic susceptibility test of isolated mutants was evaluated. DNA and RNA sequencing and real-time quantitative PCR (qPCR) were performed to identify the underlying mechanisms. The results showed metformin concentrations ranging from 10-6 to 200 mg/L caused multiple-antibiotic resistance in E. coli. After 1 day exposure to metformin at 1 ng/L, the mutation frequency in E. coli increased to 1.24 × 10-8, and it further increased to 7.13 × 10-8 when prolonged to 5 days. And the mutants showed multiple-antibiotic resistance. Whole-genome DNA analysis of mutants showed chromosome mutagenesis in marR, tonB, and fhuA. Global transcriptional analysis and qPCR revealed the expressions of emrK, emrY, cusB, cusC, hycA, cecR, marA, acrA, and acrB were upregulated and those of tonB and fhuA were significantly downregulated. Thus, an increase in efflux systems AcrAB-TolC, EmrKY-TolC, and CusCFBA together with a decrease in FhuA-TonB protein complex play vital roles in the multiple-antibiotic resistance induced by metformin.