Antibiotic resistance genes and intI1 prevalence in a swine wastewater treatment plant and correlation with metal resistance, bacterial community and wastewater parameters.

The livestock wastewater treatment plant represents an important reservoir of antibiotic resistance determinants in the environment. The study explored the prevalence of five antibiotic resistance genes (ARGs, including sulI, tetA, qnrD, mphB and mcr-1) and class 1 integron (intI1) in a typical livestock wastewater treatment plant, and analyzed their integrated association with two metal resistance genes (copA and czcA), two pathogens genes (Staphylococcus and Campylobacter), bacterial community and wastewater properties. Results indicated that all investigated genes were detected in the plant. The treatment plant could not completely remove ARGs abundances, with up to 2.2 × 104~3.7 × 108 copies/L of them remaining in the effluent. Mcr-1 was further enriched by 27-fold in the subsequent pond. The correlation analysis showed that mphB significantly correlateed with tetA and intI. Mcr-1 strongly correlated with copA. MphB and intI significantly correlated with czcA. The correlations implied a potential co-selection risk of bacterial resistant to antibiotics and metals. Redundancy analyses indicated that qnrD and mcr-1 strongly correlated with 13 and 14 bacterial genera, respectively. Most ARGs positively correlated to wastewater nutrients, indicating that an efficient reduction of wastewater nutrients would contribute to the antibiotic resistance control. The study will provide useful implications on fates and reductions of ARGs in livestock facilities and receiving environments.

Distinguishing effects of ultraviolet exposure and chlorination on the horizontal transfer of antibiotic resistance genes in municipal wastewater.

Growing attention has been paid to the dissemination of antibiotic resistance genes (ARGs) in wastewater microbial communities; however, the disinfection processes, as microbial control technologies, have not been evaluated for their impacts on ARGs transfer. In this study, the effects of ultraviolet (UV) disinfection and chlorination on the frequency of ARGs transfer have been explored based on the conjugative transfer model between Gram-negative strains of E. coli. The results indicated that UV disinfection and chlorination exhibit distinct influences on the conjugative transfer. Low UV doses (up to 8 mJ/cm2) had little influence on the frequency of conjugative transfer, and UV exposure only decreased the bacterial number but did not change the cell permeability. By comparison, low chlorine doses (up to 40 mg Cl min/L) significantly promoted the frequency of conjugative transfer by 2-5-fold. The generated chloramine stimulated the bacteria and improved the cell permeability. More pilus were induced on the surface of conjugative cells, which acted as pathways for ARGs transfer. The frequency of ARG transfers was greatly suppressed by high doses of UV (>10 mJ/cm2) or chlorine (>80 mg Cl min/L).

Fates of antibiotic resistance genes in a distributed swine wastewater treatment plant.

This study explores the prevalence, emission, and reduction of five ARGs (sulI, tetA, mphB, qnrD, and mcr-1) and integron (intI) through a distributed swine wastewater purification facility and the effluent-receiving environment. Typical metal resistance genes (MRGs), pathogenic bacterial indicators, the bacterial community, and wastewater properties were also explored to determine their effects on the fates of ARGs. Results indicated that the purification process could hardly effectively remove ARGs' prevalence. 3.1 × 104 -7.1 × 108  copies/L were present after purification, and 4%-57% of them persisted in the subsequent creek and adjacent soil. 16S rRNA sequencing suggested that the discharge of wastewater significantly changed the bacterial community in receiving creek and soil. Molecular ecological networks analysis detected the wide co-occurrence among ARGs, MRGs, and PBGs, which could further facilitate the propagation of antibiotic resistance. ARG incidence and specific bacterial genera were closely correlated, suggesting an extensive hosting relationship. Redundancy analyses showed wastewater organics and nutrients showed positive correlation to most ARGs' abundance, but negatively correlated to their relative abundance. PRACTITIONER POINTS: Fate of five ARGs and intI was studied in a swine wastewater treatment system. The treatment process could not effectively reduce ARGs' abundance. ARGs and pathogens in wastewater were transferred to the receiving creek and soil. The network analysis found wide co-occurrence among ARGs, metal resistance genes, and pathogens. Wastewater nutrients positively correlated to ARG's abundance but negatively correlated to their relative abundance.

Redistribution of intracellular and extracellular free & adsorbed antibiotic resistance genes through a wastewater treatment plant by an enhanced extracellular DNA extraction method with magnetic beads.

Due to the limitations of current extraction methods, extracellular DNA (eDNA) is rarely discerned from intracellular DNA (iDNA) despite having unique contributions to antibiotic resistance genes (ARGs) propagation. Furthermore, eDNA may be free (f-eDNA) or adsorbed to or suspended solids, including cells (a-eDNA), which affects ARG persistence and transmissivity. We developed a novel method using magnetic beads to separate iDNA, a-eDNA, and f-eDNA to assess how these physical states of ARGs change across a wastewater treatment plant. This method efficiently extracted eDNA (>85.3%) with higher recovery than current methods such as alcohol precipitation, CTAB-based extraction, and DNA extraction kits (<10%). Biological treatment and UV disinfection decreased the concentration of intracellular ARGs (iARGs) and adsorbed extracellular ARGs (a-eARGs), causing an increase of released free extracellular ARGs (f-eARGs). More ARGs were discharged through the wasted biosolids than in the effluent; iARGs and a-eARGs are prevalent in wasted biosolids ((73.9 ±â€¯22.5) % and (23.4 ±â€¯15.3) % of total ARGs respectively), while f-eARGs were prevalent in the effluent ((90.3 ±â€¯16.5) %). Bacterial community analysis showed significant correlations between specific genera and ARGs (e.g., Aeromonas, Pseudomonas and Acinetobacter were strongly correlated with multidrug-resistance gene blaTEM). This treatment system decreased the discharge of iARGs to receiving environments, however, increased eARG concentrations were present in the effluent, which may contribute to the environmental resistome.

Occurrence of super antibiotic resistance genes in the downstream of the Yangtze River in China: Prevalence and antibiotic resistance profiles.

The super antibiotic resistance genes (SARGs) demonstrate more severe threats than other antibiotic resistance genes while have not received enough attention in the environment. The study explored the prevalence and the antibiotic tolerance profiles of two typical SARGs, MCR-1 and NDM-1, and their hosting bacteria in the downstream of the Yangtze River and the nearby wastewater treatment plant (WWTP) and drinking water treatment plant (DWTP). Results indicated that MCR-1 and NDM-1 were prevalent in the influent and biological units of the WWTP. Their hosting bacteria were effectively removed, but 2.49 × 108 copies/L MCR-1 and 7.00 × 106 copies/L NDM-1 were still persistent in the effluent. In the Yangtze River, MCR-1 and NDM-1 were detected with higher abundance and antibiotic tolerance than the WWTP effluent and were significantly affected by nearby water contamination and human activities. In the DWTP, MCR-1 and NDM-1 were detected with average values 5.56 × 107 copies/L and 2.14 × 105 copies/L in the influent. Their hosting bacteria were undetectable in the effluent, but the two SARGs were still persistent with 1.39 × 107 copies/L and 6.29 × 104 copies/L, and were greatly enriched in the sludge. Molecular ecological networks demonstrated wide hosting relationships between MCR-1/NDM-1 and bacteria community in the DWTP. Redundancy analysis found that MCR-1 positively correlated with COD and NH3-N, while negatively correlated with turbidity. Additionally, MCR-1 hosting bacteria positively correlated with NO3--N and negatively correlated with COD and NH3-N. NDM-1 positively correlated with turbidity and NDM-1 hosting bacteria positively correlated with COD and NO2--N. The study demonstrated that the WWTP could not effectively remove SARGs with high amount of them being discharged into the Yangtze River. Then they were transported into the DWTP and the persistent SARGs in the effluent would probably be transferred into human, thus imposing great threats on public health.

Reductions of bacterial antibiotic resistance through five biological treatment processes treated municipal wastewater.

Wastewater treatment plants are hot spots for antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). However, limited studies have been conducted to compare the reductions of ARB and ARGs by various biological treatment processes. The study explored the reductions of heterotrophic bacteria resistant to six groups of antibiotics (vancomycin, gentamicin, erythromycin, cephalexin, tetracycline, and sulfadiazine) and corresponding resistance genes (vanA, aacC1, ereA, ampC, tetA, and sulI) by five bench-scale biological reactors. Results demonstrated that membrane bioreactor (MBR) and sequencing batch reactor (SBR) significantly reduced ARB abundances in the ranges of 2.80∼3.54 log and 2.70∼3.13 log, respectively, followed by activated sludge (AS). Biological filter (BF) and anaerobic (upflow anaerobic sludge blanket, UASB) techniques led to relatively low reductions. In contrast, ARGs were not equally reduced as ARB. AS and SBR also showed significant potentials on ARGs reduction, whilst MBR and UASB could not reduce ARGs effectively. Redundancy analysis implied that the purification of wastewater quality parameters (COD, NH4 (+)-N, and turbidity) performed a positive correlation to ARB and ARGs reductions.

Insights into the amplification of bacterial resistance to erythromycin in activated sludge.

Wastewater treatment plants are significant reservoirs for antimicrobial resistance. However, little is known about wastewater treatment effects on the variation of antibiotic resistance. The shifts of bacterial resistance to erythromycin, a macrolide widely used in human medicine, on a lab-scale activated sludge system fed with real wastewater was investigated from levels of bacteria, community and genes, in this study. The resistance variation of total heterotrophic bacteria was studied during the biological treatment process, based on culture dependent method. The alterations of bacterial community resistant to erythromycin and nine typical erythromycin resistance genes were explored with molecular approaches, including high-throughput sequencing and quantitative polymerase chain reaction. The results revealed that the total heterotrophs tolerance level to erythromycin concentrations (higher than 32 mg/L) was significantly amplified during the activated sludge treatment, with the prevalence increased from 9.6% to 21.8%. High-throughput sequencing results demonstrated an obvious increase of the total heterotrophic bacterial diversity resistant to erythromycin. Proteobacteria and Bacteroidetes were the two dominant phyla in the influent and effluent of the bioreactor. However, the prevalence of Proteobacteria decreased from 76% to 59% while the total phyla number increased greatly from 18 to 29 through activated sludge treatment. The gene proportions of erm(A), mef(E) and erm(D) were greatly amplified after biological treatment. It is proposed that the transfer of antibiotic resistance genes through the variable mixtures of bacteria in the activated sludge might be the reason for the antibiotic resistance amplification. The amplified risk of antibiotic resistance in wastewater treatment needs to be paid more attention.

The sludge loading rate regulates the growth and release of heterotrophic bacteria resistant to six types of antibiotics in wastewater activated sludge.

Wastewater treatment plants are considered as hot reservoirs of antimicrobial resistance. However, the fates of antibiotic-resistant bacteria during biological treatment processes and relevant influencing factors have not been fully understood. This study evaluated the effects of the sludge loading rate on the growth and release of six kinds of antibiotic-resistant bacteria in an activated sludge system. The results indicated that higher sludge loading rates amplified the growth of all six types of antibiotic resistant bacteria. The release of most antibiotic-resistant bacteria through both the effluent and biosolids was amplified with increased sludge loading rate. Biosolids were the main pattern for all antibiotic-resistant bacteria release in an activated sludge system, which was determined primarily by their growth in the activated sludge. A higher sludge loading rate reactor tended to retain more antibiotic resistance. An activated sludge system with lower sludge loading rates was considered more conducive to the control of antibiotic resistance.

Fate of antibiotic resistant bacteria and genes during wastewater chlorination: implication for antibiotic resistance control.

This study investigated fates of nine antibiotic-resistant bacteria as well as two series of antibiotic resistance genes in wastewater treated by various doses of chlorine (0, 15, 30, 60, 150 and 300 mg Cl2 min/L). The results indicated that chlorination was effective in inactivating antibiotic-resistant bacteria. Most bacteria were inactivated completely at the lowest dose (15 mg Cl2 min/L). By comparison, sulfadiazine- and erythromycin-resistant bacteria exhibited tolerance to low chlorine dose (up to 60 mg Cl2 min/L). However, quantitative real-time PCRs revealed that chlorination decreased limited erythromycin or tetracycline resistance genes, with the removal levels of overall erythromycin and tetracycline resistance genes at 0.42 ± 0.12 log and 0.10 ± 0.02 log, respectively. About 40% of erythromycin-resistance genes and 80% of tetracycline resistance genes could not be removed by chlorination. Chlorination was considered not effective in controlling antimicrobial resistance. More concern needs to be paid to the potential risk of antibiotic resistance genes in the wastewater after chlorination.

Monitoring and assessing the impact of wastewater treatment on release of both antibiotic-resistant bacteria and their typical genes in a Chinese municipal wastewater treatment plant.

Wastewater treatment plants (WWTPs) are important hotspots for the spread of antibiotic resistance. However, the release and impact factors of both antibiotic resistant bacteria and the relevant genes over long periods in WWTPs have rarely been investigated. In this study, the fate of bacteria and genes resistant to six commonly used antibiotics was assessed over a whole year. In WWTP effluent and biosolids, a high prevalence of heterotrophic bacteria resistant to vancomycin, cephalexin, sulfadiazine and erythromycin were detected, each with a proportion of over 30%. The corresponding genes (vanA, ampC, sulI and ereA) were all detected in proportions of (2.2 ± 0.8) × 10(-10), (6.2 ± 3.2) × 10(-9), (1.2 ± 0.8) × 10(-7) and (7.6 ± 4.8) × 10(-8), respectively, in the effluent. The sampling season imposed considerable influence on the release of all ARB. High release loads of most ARB were detected in the spring, while low release loads were generally found in the winter. In comparison, the ARG loads changed only slightly over various seasons. No statistical relevance was found between all ARB abundances and their corresponding genes over the long-term investigation period. This inconsistent behavior indicates that bacteria and genes should both be considered when exploring resistance characteristics in wastewater. A redundancy analysis was adopted to assess the impact of wastewater quality and operational conditions on antibiotic resistance. The results indicated that most ARB and ARG proportions were positively related to the COD and turbidity of the raw sewage, while negatively related to those of the effluent. DO and temperature exhibited strong negative relevance to most ARB prevalence.

Microbial selectivity of UV treatment on antibiotic-resistant heterotrophic bacteria in secondary effluents of a municipal wastewater treatment plant.

Little is known about the microbial selectivity of UV treatment for antibiotic resistant bacteria, and the results of limited studies are conflicting. To understand the effect of UV disinfection on antibiotic resistant bacteria, both total heterotrophic bacteria and antibiotic resistant bacteria (including cephalexin-, ciprofloxacin-, erythromycin-, gentamicin-, vancomycin-, sulfadiazine-, rifampicin-, tetracycline- and chloramphenicol-resistant bacteria) were examined in secondary effluent samples from a municipal wastewater treatment plant. Bacteria resistant to both erythromycin and tetracycline were chosen as the representative of multiple-antibiotic-resistant bacteria and their characteristics after UV treatment were also investigated. UV disinfection results in effective inactivation for total heterotrophic bacteria, as well as all antibiotic resistant bacteria. After UV treatment at a fluence of 5 mJ/cm(2), the log reductions of nine types of antibiotic resistant bacteria varied from 1.0 ± 0.1 to 2.4 ± 0.1. Bacteria resistant to both erythromycin and tetracycline had a similar fluence response as did total heterotrophic bacteria. The findings suggest that UV disinfection could eliminate antibiotic resistance in wastewater treatment effluents and thus ensure public health security. Our experimental results indicated that UV disinfection led to enrichment of bacteria with resistance to sulfadiazine, vancomycin, rifampicin, tetracycline and chloramphenicol, while the proportions of cephalexin-, erythromycin-, gentamicin- and ciprofloxacin-resistant bacteria in the wastewater decreased. This reveals the microbial selectivity of UV disinfection for antibiotic resistant bacteria.

Ultraviolet reduction of erythromycin and tetracycline resistant heterotrophic bacteria and their resistance genes in municipal wastewater.

Antibiotic resistance in wastewater is becoming a major public health concern, but poorly understood about impact of disinfection on antibiotic resistant bacteria and antibiotic resistance genes. The UV disinfection of antibiotic resistant heterotrophic bacteria and their relevant genes in the wastewater of a municipal wastewater treatment plant has been evaluated. Two commonly used antibiotics, erythromycin and tetracycline were selected because of their wide occurrences in regard to the antibiotic resistance problem. After UV treatment at a fluence of 5mJcm(-2), the log reductions of heterotrophic bacteria resistant to erythromycin and tetracycline in the wastewater were found to be 1.4±0.1 and 1.1±0.1, respectively. The proportion of tetracycline-resistant bacteria (5%) was nearly double of that before UV disinfection (3%). Tetracycline-resistant bacteria exhibited more tolerance to UV irradiation compared to the erythromycin-resistant bacteria (p<0.05). Gene copy numbers were quantified via qPCR and normalized to the volume of original sample. The total concentrations of erythromycin- and tetracycline-resistance genes were (3.6±0.2)×10(5) and (2.5±0.1)×10(5) copies L(-1), respectively. UV treatment at a fluence of 5mJcm(-2) removed the total erythromycin- and tetracycline-resistance genes by 3.0±0.1 log and 1.9±0.1 log, respectively. UV treatment was effective in reducing antibiotic resistance in the wastewater.