Deciphering Multidrug-Resistant Plasmids in Disinfection Residual Bacteria from a Wastewater Treatment Plant.

Current disinfection processes pose an emerging environmental risk due to the ineffective removal of antibiotic-resistant bacteria, especially disinfection residual bacteria (DRB) carrying multidrug-resistant plasmids (MRPs). However, the characteristics of DRB-carried MRPs are poorly understood. In this study, qPCR analysis reveals that the total absolute abundance of four plasmids in postdisinfection effluent decreases by 1.15 log units, while their relative abundance increases by 0.11 copies/cell compared to investigated wastewater treatment plant (WWTP) influent. We obtain three distinctive DRB-carried MRPs (pWWTP-01-03) from postdisinfection effluent, each carrying 9-11 antibiotic-resistant genes (ARGs). pWWTP-01 contains all 11 ARGs within an ∼25 Kbp chimeric genomic island showing strong patterns of recombination with MRPs from foodborne outbreaks and hospitals. Antibiotic-, disinfectant-, and heavy-metal-resistant genes on the same plasmid underscore the potential roles of disinfectants and heavy metals in the coselection of ARGs. Additionally, pWWTP-02 harbors an adhesin-type virulence operon, implying risks of both antibiotic resistance and pathogenicity upon entering environments. Furthermore, some MRPs from DRB are capable of transferring and could confer selective advantages to recipients under environmentally relevant antibiotic pressure. Overall, this study advances our understanding of DRB-carried MRPs and highlights the imminent need to monitor and control wastewater MRPs for environmental security.

Comprehensive profiling and risk assessment of antibiotic resistance genes in a drinking water watershed by integrated analysis of air-water-soil.

The prevalence of antibiotic resistance genes (ARGs) in diverse habitats threatens public health. Watersheds represent critical freshwater ecosystems that interact with both the soil and atmosphere. However, a holistic understanding of ARGs distribution across these environmental media is currently inadequate. We profiled ARGs and bacterial communities in air-water-soil in the same watershed area during four seasons using high-throughput qPCR and 16S rRNA gene sequencing. Our findings demonstrated that aminoglycoside resistance genes (58.5%) were dominant in water, and multidrug resistance genes (55.2% and 54.2%) were dominant in soil and air. Five ARGs and nineteen bacterial genera were consistently detected in all samples, were named as shared genes or bacteria. Co-occurrence Network analysis revealed the co-occurrence module of resistance genes, mobile genetic elements (MGEs), and potential bacterial hosts, indicating that shared genes and bacteria may persist and co-spread across different environmental media. The risk assessment framework, based on ARGs' abundance, detection rate, and mobility, identified 33 high-risk ARGs. This is essential to evaluate the health risks of ARGs and to develop strategies to limit the threat of antibiotic resistance. Our study offers new insights into the risks associated with ARGs in the environment and suggests that ARGs may depend on specific bacterial cohabitants that co-exist with MGEs to facilitate their spread across environmental interfaces.

Microplastics exacerbate co-occurrence and horizontal transfer of antibiotic resistance genes.

Microplastic pollution is a rising environmental issue worldwide. Microplastics can provide a niche for the microbiome, especially for antibiotic-resistant bacteria, which could increase the transmission of antibiotic resistance genes (ARGs). However, the interactions between microplastics and ARGs are still indistinct in environmental settings. Microplastics were found to be significantly correlated with ARGs (p < 0.001), based on the analysis of samples taken from a chicken farm and its surrounding farmlands. Analysis of chicken feces revealed the highest abundance of microplastics (14.9 items/g) and ARGs (6.24 ×108 copies/g), suggesting that chicken farms could be the hotspot for the co-spread of microplastics and ARGs. Conjugative transfer experiments were performed to investigate the effects of microplastic exposure for different concentrations and sizes on the horizontal gene transfer (HGT) of ARGs between bacteria. Results showed that the microplastics significantly enhanced the bacterial conjugative transfer frequency by 1.4-1.7 folds indicating that microplastics could aggravate ARG dissemination in the environment. Potential mechanisms related to the up-regulation of rpoS, ompA, ompC, ompF, trbBp, traF, trfAp, traJ, and down-regulation of korA, korB, and trbA were induced by microplastics. These findings highlighted the co-occurrence of microplastics and ARGs in the agricultural environment and the exacerbation of ARGs' prevalence via rising the HGT derived from microplastics.

Deciphering basic and key traits of antibiotic resistome in influent and effluent of hospital wastewater treatment systems.

Hospital wastewater treatment system (HWTS) is an important source and environmental reservoir of clinically relevant antibiotic resistance genes (ARGs). However, how antibiotic resistome of clinical wastewater changed in HWTS is poorly understood. Herein, the basic quantitative traits (i.e., diversity and abundance) of ARGs in three HWTSs were profiled by metagenomics. In total, 709 ARG subtypes belonging to 20 ARG types were detected with relative abundance ranging from 1.12 × 10-5 to 7.33 × 10-1 copies/cell. Notably, most ARGs could not be significantly removed by chlorination treatment in the HWTS. These ARGs were identified to confer resistance to almost all major classes of antibiotics and include ARGs of last-resort antibiotics, such as blaNDM, mcr and tet(X) which were abundantly occurred in HWTS with 19, 5 and 7 variants, respectively. Moreover, qualitative analysis based on metagenome-assembled genome (MAG) analysis revealed that the putative hosts of the identified ARGs were broadly distributed into at least 8 dominant bacterial phyla. Of the 107 ARG-carrying MAGs recovered, 39 encoded multi-antibiotic resistance and 16 belonged to antibiotic resistant pathogens. Further analysis of co-occurrence patterns of ARGs with mobile genetic elements suggested their potential mobility. These key qualitative traits of ARGs provided further information about their phylogeny and genetic context. This study sheds light on the key traits of ARGs associated with resistance dissemination and pathogenicity and health risks of clinical wastewater.

Simultaneously screening for methicillin-resistant Staphylococcus aureus and its susceptibility to potentiated penicillins.

Introduction. We recently revealed that a significant proportion of clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates are susceptible to pencillins and clavulanic acid (potentiated penicillins), including widely available combinations such as co-amoxiclav. These isolates also showed increased susceptibility to oxacillin on Iso-Sensitest Agar (ISA).Hypothesis/Gap Statement. The increased susceptibility to oxacillin displayed on ISA by these MRSA isolates may be used to distinguish them from the resistant ones.Aim. We aimed to develop a method to simultaneously screen a S. aureus clinical isolate for its susceptibility to methicillin and potentiated penicillins.Methodology. A double-disc diffusion method using 10 µg cefoxitin and 1 µg oxacillin discs on ISA was developed and tested against a panel of 120 whole genome-sequenced MRSA isolates. The sensitivity of the method was compared with that of previously published genotypic and phenotypic methods. In addition, double-disc diffusion was performed for all isolates on Müller-Hinton agar (MHA) following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) protocol.Results. All isolates (120/120) were reconfirmed to be phenotypically MRSA, as indicated by the result of cefoxitin disc diffusion testing. All isolates (40/40) that had a pencillins and clavulanic acid (Pen-Clav)-resistant genotype were not inhibited by oxacillin, while 77/80 (96.3 %) isolates that had a Pen-Clav-susceptible genotype were inhibited by oxacillin on ISA. The results also showed that the EUCAST method using MHA correctly identified all isolates as MRSA but failed to distinguish the Pen-Clav-susceptible isolates from the Pen-Clav-resistant isolates.Conclusions. This double-disc diffusion method using ISA could be used to accurately screen for clinical MRSA isolates and determine their susceptibility to Pen-Clav simultaneously, rapidly identifying MRSA infections that might be suitable for treatment with potentiated penicillins.

Mechanism and potential risk of antibiotic resistant bacteria carrying last resort antibiotic resistance genes under electrochemical treatment.

The significant rise in the number of antibiotic resistance genes (ARGs) that resulted from our abuse of antibiotics could do severe harm to public health as well as to the environment. We investigated removal efficiency and removal mechanism of electrochemical (EC) treatment based on 6 different bacteria isolated from hospital wastewater carrying 3 last resort ARGs including NDM-1, mcr-1 and tetX respectively. We found that the removal efficiency of ARGs increased with the increase of both voltage and electrolysis time while the maximum removal efficiency can reach 90%. The optimal treatment voltage and treatment time were 3 V and 120 min, respectively. Temperature, pH and other factors had little influence on the EC treatment process. The mechanism of EC treatment was explored from the macroscopic and microscopic levels by scanning electron microscopy (SEM) and flow cytometry. Our results showed that EC treatment significantly changed the permeability of cell membrane and caused cells successively experience early cell apoptosis, late cell apoptosis and cell necrosis. Moreover, compared with traditional disinfection methods, EC treatment had less potential risks. The conjugative transfer frequencies of cells were significantly reduced after treatment. Less than 1% of bacteria entered the viable but nonculturable (VBNC) state and less than 5% of intracellular ARGs (iARGs) turned into extracellular ARGs (eARGs). Our findings provide new insights into as well as important reference for future electrochemical treatment in removing ARB from hospital wastewater.

Landscape of genes in hospital wastewater breaking through the defense line of last-resort antibiotics.

Hospital wastewater contains abundant antibiotics, antibiotic resistance genes (ARGs), and pathogens. Last-resort antibiotic resistance genes (LARGs) include the New Delhi metallo-ß-lactamase gene blaNDM, mobile colistin resistance gene mcr and tigecycline resistance gene tet(X) which confers resistance to carbapenems, colistin and tigecycline. The presence and significance of LARGs in hospital wastewater treatment systems (HWTS) have not yet been systematically explored. Here, LARG variants were shown to be prevalent both influents and effluents of HWTS. A total of 989 Enterobacteriaceae isolates that confer resistance to last-resort antibiotics were collected from effluents and multiple genetic contexts of LARGs were analyzed. LARGs-carrying plasmids were confirmed to show high multidrug phenotypes and transferability. We also discovered the co-occurrence of plasmids harboring blaNDM-1 and mcr-1 in single Escherichia coli, as well as E. coli HM016 containing two unique mcr-1-carrying plasmids. This result might accelerate co-dissemination of LARGs under environmental selection pressure. Different core genetic arrangements in these strains suggest several evolutionary pathways in HWTS. The resistance functions of LARGs were confirmed in vitro and in vivo by mass spectrometry. This study provides novel insights into the diversity, genetic context and function of critical ARGs in HWTS. The results raise the concern that LARGs may further spread into the environment, thus, more stringent discharge standards and regulations for hospital wastewater are urgently needed.

Spread of antibiotic resistance genes and microbiota in airborne particulate matter, dust, and human airways in the urban hospital.

Antimicrobial resistance is an increasingly serious threat to public health worldwide. The presence of antibiotic resistance genes (ARGs) in human airways and relevant environments has not received significant attention. In this study, abundances of ARGs and microbes from airborne particulate matter, dust, and human airways in a hospital were profiled using high-throughput qPCR and 16S rRNA gene sequencing. More diverse ARGs and microbes in indoor dust and higher levels of ARGs in particulate matter PM10 and PM2.5 were observed. Macrolides and aminoglycoside resistance genes were the most abundant ARGs in the airway and environmental samples, respectively. Moreover, the co-occurrences of priority pathogens, ARGs, and mobile genetic elements (MGEs) were shown by the Network analysis. Campylobacter spp. and Staphylococcus spp. positively correlated with fluoroquinolone (vatC-02, mexD) and ß-lactams (blaZ, mecA) resistance genes, respectively. In this regard, based on SourceTracker analysis, inhalable particles contributed to 4.0% to 5.5% of ARGs in human airway samples, suggesting an important exchange between airborne inhalable particles and human commensals. This study may advance knowledge about ARGs in airborne particulate matter and dust associated environments, reveal their potential link between environments and humans, and provide a new sight and fundamental data for ARG risk assessment.

Temporal variation and sharing of antibiotic resistance genes between water and wild fish gut in a peri-urban river.

Antibiotic resistance genes (ARGs) as emergence contaminations have spread widely in the water environment. Wild fish may be recipients and communicators of ARGs in the water environment, however, the distribution and transmission of ARGs in the wild fish and relevant water environment were rarely reported. Here, we have profiled ARGs and bacterial communities in wild freshwater fish and relevant water in a peri-urban river using high-throughput qPCR and 16S rRNA gene sequence. A total of 80 and 220 unique ARG subtypes were identified in fish and water samples. Fish and water both showed significant ARG seasonal variations (P < 0.05). The highest absolute abundance of ARGs in fish and water occurred in summer (1.32 × 109 copies per g, on average) and autumn (9.04 × 106 copies per mL), respectively. In addition, the bipartite network analysis showed that 9 ARGs and 1 mobile genetic element continuously shared in fish and water. Furthermore, bacteria shared in fish and water were found to significantly correlate with shard ARGs. The findings demonstrate that bacteria and ARGs in fish and water could interconnect and ARGs might transfer between fish and water using bacteria as a spreading medium.

Characteristics of bacterial community and ARGs profile in engineered goldfish tanks with stresses of sulfanilamide and copper.

Abuse of antibiotics in aquaculture have been alarming and might aggravate spread of resistance genes in the environment. Holistic ARGs proliferation checks require deeper analyses of coupled absolute abundances in 16S rRNA bacteria communities at the phylum level to detect biomarkers. Sulfanilamide (sul) and copper II sulfate (CuSO4 II) were, therefore, designed and added as separate or combined treatments in 9 replicate engineered goldfish tanks comprising 3 individual sul, 3 CuSO4 II, 3 (sul + CuSO4 II) combinations, and 3 controls within 180 days. The DNA from water and fish guts was sequenced under qPCR to determine 16S rRNA bacteria biomarkers co-occurring with the correspondent ARGs. Combined chemical addition at 0.8-1.5 mg sul + 0.5-1.0 mg CuSO4 II/3 L of tank waters reduced sequenced 16S rRNA bacteria absolute abundances in fish gut and water samples while portraying the biomarkers. Absolute abundances of the entire 16S rRNA bacteria was higher in fish guts (3.4 × 1014-4.9 × 108 copies/g) than water samples (1.5 × 109-2.6 × 1015 copies/L), respectively. Much as sul 1(log) were dominant over intl 1(log) genes, and their fundamental profiles were also higher in the fish guts than water samples; the Spearman's correlation analyses revealed positive relationship (p < 0.01 and r = 0.873) among the biomarkers of both ARG pairs at the phylum level and the physicochemical parameters. In the fish gut and water samples ratios, Bacteroidetes (10-85:12-85%) > Proteobacteria (10-50:15-65%) > Planktomycetes (10-52:8-25%) featured prominently based on LEfSe use as the hot-spotted biomarkers, hence justifying its higher prospects towards innovative environmental microbiological and biotechnological studies.

Behavior of antibiotic resistance genes in a wastewater treatment plant with different upgrading processes.

Wastewater treatment plants (WWTPs) in China have been upgraded or renovated with a variety of emerging processes, but a comprehensive understanding of the behavior of antibiotic resistance genes (ARGs) in these WWTPs is still lacking. Here, the distribution of ARGs and bacterial community were investigated in a wastewater treatment plant with upgrading processes (WWTP-UP). 238 unique ARGs were detected in all samples. During the study period, the average ARGs concentration decreased by 98.4% along the entire treatment process. The removal efficiency of A2/O-membrane bioreactor (MBR) process was significantly higher than that of A2/O-high efficiency flocculent settling/cloth media filter (HEFS/CMF) process (p < 0.05), which corresponded to 3.5 and 2.1 log values on average, respectively. Notably, 35 ARGs and 14 mobile genetic elements (MGEs) were persistent in all samples. Based on the co-occurrence pattern revealed by network analysis, persistent ARGs possibly spread through the transfer of persistent MGEs among persistent bacteria. Using multiple linear regression analysis, we obtained 3 to 5 possible indicators for major ARG types, which might be served to evaluate the general distribution of ARGs or even predict the abundance of different ARG types. Our findings provide new insights into the impacts of upgrading process on ARGs and highlight the need for better strategies to improve ARGs elimination in WWTPs.

Prevalence of multi-resistant plasmids in hospital inhalable particulate matter (PM) and its impact on horizontal gene transfer.

Antibiotic resistance is exacerbated by the exchange of antibiotic resistance genes (ARGs) between microbes from diverse habitats. Plasmids are important ARGs mobile elements and are spread by horizontal gene transfer (HGT). In this study, we demonstrated the presence of multi-resistant plasmids from inhalable particulate matter (PM) and its effect on gene horizontal transfer. Three transferable multi-resistant plasmids were identified from PM in a hospital, using conjugative mating assays and nanopore sequencing. pTAir-3 contained 26 horizontal transfer elements and 10 ARGs. Importantly pTAir-5 harbored carbapenem resistance gene (blaOXA) which shows homology to plasmids from human and pig commensal bacteria, thus indicating that PM is a media for antibiotic resistant plasmid spread. In addition, 125 µg/mL PM2.5 and PM10 significantly increased the conjugative transfer rate by 110% and 30%, respectively, and augmented reactive oxygen species (ROS) levels. Underlying mechanisms were revealed by identifying the upregulated expressional levels of genes related to ROS, SOS, cell membranes, pilus generation, and transposition via genome-wide RNA sequencing. The study highlights the airborne spread of multi-resistant plasmids and the impact of inhalable PM on the horizontal transfer of antibiotic resistance.

The impact and mechanism of quaternary ammonium compounds on the transmission of antibiotic resistance genes.

The emergence of antibiotic resistance genes (ARGs) in microbes can be largely attributed to the abuse and misuse of antibiotics and biocides. Quaternary ammonium compounds (QACs) have been used worldwide as common disinfectants and detergents; however, their potential impact on the spread and diffusion of ARGs is still unknown. In this study, we detected the QAC resistance gene (qacEΔ1), the 1 integron gene (intI1), and 12 ARGs (sul1, sul2, cfr, cml, fexA, tetA, tetG, tetQ, tetX, ermB, blaTEM, and dfrA1) in 48 water samples from three watersheds by quantitative PCR (qPCR). We investigated the evolution of bacterial antibiotic resistance under QAC and antibiotic environmental pressures by long-term continuous culture. In addition, five QACs were selected to investigate the effect of QAC on the efficiency of conjugation transfer. The changes in bacterial cell membrane and production of reactive oxygen species (ROS) were detected by flow cytometry, revealing the mechanism by which QAC affects the spread of antibiotic resistance. Our results showed that the QAC resistance gene was ubiquitous in watersheds and it had significant correlation with intI1 and seven ARGs (r = 0.999, p < 0.01). QACs could increase the resistance of bacteria to multiple antibiotics. Furthermore, all five QACs promoted the conjugation transfer of the RP4 plasmid; the optimal concentration of QACs was about 10-1-10-2 mg/L and their transfer efficiencies were between 1.33 × 10-6 and 8.87 × 10-5. QACs enhanced membrane permeability of bacterial cells and stimulated bacteria to produce ROS, which potentially promoted the transfer of plasmids between bacteria. In conclusion, this study demonstrated that QACs may facilitate the evolution and gene transfer of antibiotic resistance gene among microbiome.

Prevalence and transmission of antibiotic resistance and microbiota between humans and water environments.

The transmission routes for antibiotic resistance genes (ARGs) and microbiota between humans and water environments is poorly characterized. Here, we used high-throughput qPCR analyses and 16S rRNA gene sequencing to examine the occurrence and abundance of antibiotic resistance genes and microbiota in both healthy humans and associated water environments from a Chinese village. Humans carried the most diverse assemblage of ARGs, with 234 different ARGs being detected. The total abundance of ARGs in feces, on skin, and in the effluent from domestic sewage treatment systems were approximately 23, 2, and 7 times higher than their abundance in river samples. In total, 53 ARGs and 28 bacteria genera that were present in human feces could also be found in the influent and effluent of rural sewage treatment systems, and also downstream of the effluent release point. We identified the bacterial taxa that showed a significant association with ARGs (P < 0.01, r > 0.8) by network analysis, supporting the idea that these bacteria could carry some ARGs and transfer between humans and the environment. Analysis of ARGs and microbiota in humans and in water environments helps to define the transmission routes and dynamics of antibiotic resistance within these environments. This study highlights human contribution to the load of ARGs into the environment and suggests means to prevent such dissemination.

Biomarkers of antibiotic resistance genes during seasonal changes in wastewater treatment systems.

To evaluate the seasonal distribution of antibiotic resistance genes (ARGs) and explore the reason for their patterns in different seasons and different systems, two wastewater treatment systems were selected and analyzed using high-throughput qPCR. Linear discriminant analysis (LDA) effect size (LEfSe) was used to discover the differential ARGs (biomarkers) and estimate the biomarkers' effect size. We found that the total absolute abundances of ARGs in inflows and excess sludge samples had no obvious seasonal fluctuations, while those in winter outflow samples decreased in comparison with the inflow samples. Eleven differentially abundant ARGs (biomarker genes, BmGs) (aadA5-02, aac-6-II, cmlA1-01, cmlA1-02, blaOXA10-02, aadA-02, tetX, aadA1, ereA, qacEΔ1-01, and blaTEM) in summer samples and 10 BmGs (tet-32, tetA-02, aacC2, vanC-03, aac-6-I1, tetE, ermB, mefA, tnpA - 07, and sul2) in winter samples were validated. According to 16S rRNA gene sequencing, the relative abundance of bacteria at the phylum level exhibited significant seasonal changes in outflow water (OW), and biomarker bacteria (BmB) were discovered at the family (or genus) level. Synechococcus and vadinCA02 are BmB in summer, and Trichococcus, Lactococcus, Pelosinus, Janthinobacterium, Nitrosomonadaceae and Sterolibacterium are BmB in winter. In addition, BmB have good correlations with BmGs in the same season, which indicates that bacterial community changes drive different distributions of ARGs during seasonal changes and that LEfSe is an acute and effective method for finding significantly different ARGs and bacteria between two or more classes. In conclusion, this study demonstrated the seasonal changes of BmGs and BmB at two wastewater treatment systems.

Antibiotic resistance genes in an urban river as impacted by bacterial community and physicochemical parameters.

Antibiotic resistance genes (ARGs) in urban rivers are a serious public health concern in regions with poorly planned, rapid development. To gain insights into the predominant factors affecting the fate of ARGs in a highly polluted urban river in eastern China, a total of 285 ARGs, microbial communities, and 20 physicochemical parameters were analyzed for 17 sites. A total of 258 unique ARGs were detected using high-throughput qPCR, and the absolute abundance of total ARGs was positively correlated with total organic carbon and total dissolved nitrogen concentrations (P < 0.01). ARG abundance and diversity were greatly altered by microbial community structure. Variation partitioning analysis showed that the combined effects of multiple factors contributed to the profile and dissemination of ARGs, and variation of microbial communities was the major factor affecting the distribution of ARGs. The disparate distribution of some bacteria, including Bacteroides from mammalian gastrointestinal flora, Burkholderia from zoonotic infectious diseases, and Zoogloea from wastewater treatment, indicates that the urban river was strongly influenced by point-source pollution. Results imply that microbial community shifts caused by changes in water quality may lead to the spread of ARGs, and point-source pollution in urban rivers requires greater attention to control the transfer of ARGs between environmental bacteria and pathogens.

[Reducing Effect of Three Disinfection Technologies for Sulfonamides Resistance Genes].

Antibiotic resistance genes (ARGs) in the environment have gradually engendered extensive concern recently due to the harm caused to environment and human beings. In this study, two sulfonamides resistant bacteria isolated from secondary effluents of a municipal wastewater treatment plant were used to compare the efficacy of three different disinfection processes (chlorination, ultraviolet and ozone) in reducing sulfonamides resistance pollution using serial dilution plate counting method and qPCR. The behavior characteristics of sulfonamides resistance genes during the disinfection process were elucidated using disinfection+DNaseⅠ treatment. The results showed that all three disinfection techniques could reduce the concentration of target bacteria, but the abundance of sulfonamides resistance genes was still at a high level. Furthermore, chlorination disinfection might reduce sulfonamides resistance genes by reducing microbial biomass. Ultraviolet disinfection could damage genes directly. However, during ozone disinfection, sulfonamides resistance genes would escape to environment, and its potential environmental risks could not be ignored.