Microbial communities responded to tetracyclines and Cu(II) in constructed wetlands microcosms with Myriophyllum aquaticum.

Combined antibiotic and heavy metal pollution has generated considerable concern. Constructed wetlands (CWs) have been shown to efficiently remove pollutants; however, the microbial community responses to combined pollutants remain enigmatic. In this study, seven microcosm CWs were planted with Myriophyllum aquaticum, spiked with tetracyclines (TCs) (300-30,000 µg/L), alone or with Cu(II), to investigate the response of plant-associated microbial communities. TCs and the Cu/TC ratio greatly affected the performance of CWs. Tetracyclines led to higher microbial diversity, evenness and richness, while UniFrac distances and principal coordinate (PCO) and redundancy analyses revealed that the co-presence of TCs and Cu(II) led to variations in bacterial communities. Proteobacteria, Cyanobacteria and Bacteroidetes were the dominant microbial phyla and Cloacibacterium, Hydrogenophaga, Rheinheimera and Denitratisoma accounted for 6.2-21.0% of all genera. Therefore, the co-occurrence of heavy metals should be considered when judging the removal potential of TCs in phytoremediation.

Hormetic dose-dependent response about typical antibiotics and their mixtures on plasmid conjugative transfer of Escherichia coli and its relationship with toxic effects on growth.

Bacterial resistance caused by the abuse of antibiotics has attracted worldwide attention. However, there are few studies exploring bacterial resistance under the environmental exposure condition of antibiotics that is featured by low-dose and mixture. In this study, sulfonamides (SAs), sulfonamide potentiators (SAPs) and tetracyclines (TCs) were used to determine the effects of antibiotics on plasmid RP4 conjugative transfer of Escherichia coli (E. coli) under single or combined exposure, and the relationship between the effects of antibiotics on conjugative transfer and growth was investigated. The results show that the effects of single or binary antibiotics on plasmid RP4 conjugative transfer all exhibit a hormetic phenomenon. The linear regression reveals that the concentrations of the three antibiotics promoting conjugative transfer are correlated with the concentrations promoting growth and the physicochemical properties of the compounds. The combined effects of SAs-SAPs and SAs-TCs on plasmid conjugative transfer are mainly synergistic and antagonistic. While SAPs provide more effective concentrations for the promotion of conjugative transfer in SAs-SAPs mixtures, SAs play a more important role in promoting conjugative transfer in SAs-TCs mixtures. Mechanism explanation shows that SAs, SAPs and TCs inhibit bacterial growth by acting on their target proteins DHPS, DHFR and 30S ribosomal subunit, respectively. This study indicates that toxic stress stimulates the occurrence of conjugative transfer and promotes the development of bacterial resistance, which will provide a reference for resistance risk assessment of antibiotic exposure.

Tetracyclines lead to ammonium accumulation during nitrification process.

The effect of tetracyclines used for swine food-production (tetracycline and oxytetracycline) on enriched nitrifying bacteria cultures over time was investigated in this study. Short-term exposure assays were performed in different concentrations of each antibiotic, using ammonia oxidizing bacteria (AOB) culture and nitrifying bacteria. The results pointed out a higher inhibitory effect of tetracycline on both bacterial communities. The AOB was more sensitive to antibiotic exposure when compared to the nitrifying culture. Although high antibiotic concentrations were applied, the half maximal inhibitory concentration (IC50) was achieved only for the AOB culture exposed to tetracycline at a concentration of 273 mg L-1. Nonetheless, the long-term exposure assay demonstrated a reduction of the tetracycline inhibition effect against AOB. The exposure to 100 mg L-1 of tetracycline (TC) did not show relevant influence over ammonium conversion efficiency; however, at 128 mg L-1 of TC, the efficiency decreased from 94% to 72%. Further investigation revealed that TC reduced the final effluent quality due to the development of a resistance mechanism by AOB culture against this antibiotic. This mechanism involves increasing the excretion of extracellular polymeric substances (EPS) and soluble microbial products (SMP), which probably increases BOD, and reduces ammonia consumption by the bacterial culture.

Ca2+ protect zebrafish embryos from water acidification.

Ionizable strategies are routinely used to enhance the solubility and dissolution rates of various pharmaceuticals. These chemicals may directly affect aquatic environment once discharged from factories, hospitals or livestock farms. Here, we assessed the potential side effect of tetracyclines (TCs) on the development of zebrafish embryos. Tetracycline hydrochloride decreased water pH from 6.4 to 4.4 at 30 mg/L. Acidified water exceeded the tolerance of zebrafish embryos in pure water during the early ten hours post fertilization (hpf). Interestingly, we found that Ca2+ in the embryo medium could increase the tolerance of embryos to acidified water. Furthermore, we found that the protection of Ca2+ was not due to the formation of TCs-Ca2+ complexes under acidic condition, based on spectral analysis. Meanwhile we showed that exogenous addition of Ca2+ could inhibit the accumulation of Ca2+ from the cytoplasm to the surface of embryos. These results may shed light on the strategies for protecting aquatic animals from acidic environments.

Toxic effects of tetracycline and its degradation products on freshwater green algae.

Tetracycline antibiotics are the most widely used antibiotics in the world and the most common veterinary drugs and feed additives used in livestock, poultry and aquaculture operations. Because antibiotics cannot be completely removed by currently existing sewage treatment facilities, these materials enter the environment directly via sewage treatment plant discharge, where they degrade. Accordingly, the metabolism and the ecological toxicity of tetracycline degradation products are worthy of attention. Herein, we investigated the effects of tetracycline and its degradation products (anhydrotetracycline and epitetracycline hydrochloride) on the growth, cell structure and algal cell oxidative stress of common Chlorella vulgaris. The results showed that the 96h-EC50 values of tetracycline (TC), anhydrotetracycline (ATC) and epitetracycline (ETC) on algal cells were 7.73, 5.96 and 8.42 mg/L, respectively. Moreover, the permeability of algal cells exposed to high concentrations of these three drugs was significantly enhanced. In addition, there were structural changes in the cells such as plasmolysis and starch granule deposition appeared, the thylakoid lamellae in the chloroplasts became blurred and deformed, and the vacuoles became larger. Exposure to higher concentrations (>5 mg/L) of TC and its degradation products ATC and ETC significantly upregulated the activity of ROS, as well as the antioxidants SOD and CAT. The levels of the lipid peroxidation product MDA also showed the same trend. Finally, ATC had the strongest toxicity toward algal cells, followed by TC and then ETC.

Phosphine production in anaerobic wastewater treatment under tetracycline antibiotic pressure.

The influence of tetracycline (TC) antibiotics on phosphine (PH3) production in the anaerobic wastewater treatment was studied. A lab-scale anaerobic baffled reactor with three compartments was employed to simulate this process. The reactor was operated in a TC-absence wastewater and 250µg/L TC-presence wastewater for three months after a start-up period, respectively. The responses of pH, oxidation-reduction potential (ORP), chemical oxygen demand (COD), total phosphorus (TP), enzymes activity (dehydrogenase and acid phosphatase), and microbial community were investigated to reveal the effect of TC on PH3 production. Results suggested that the dehydrogenase (DH) activity, acid phosphatase (ACP) activity and COD have positive relationship with PH3 production, while pH, ORP level and the TP in liquid phase have negative relationship with PH3 production. With prolonged TC exposure, decrease in pH and increase in DH activity are beneficial to PH3 production, while decrease in COD and ACP activity are not the limiting factors for PH3 production.

Reproductive toxicity of ß-diketone antibiotic mixtures to zebrafish (Danio rerio).

So far, few data are available on the reproductive toxicological assessment of ß-diketone antibiotics (DKAs), a class of ubiquitous pseudo-persistent pollutant, in zebrafish (Danio rerio). Herein, we reported the reproductive effects of DKAs by means of transcriptome analysis (F1-zebrafish), changes in a series of reproductive indices (F0-zebrafish) and histopathological observations. A total of 1170, 983 and 1399 genes were found to be differentially expressed when compared control vs. 6.25mg/L, control vs. 12.5mg/L and 6.25 vs. 12.5mg/L DKA-exposure treatments, respectively. Among three comparison groups, 670, 569 and 821 genes were respectively assigned for GO analyses based on matches with sequences of known functions. In 149 KEGG-noted metabolic pathways, the preferential one was the MAPK (mitogen-activated protein kinase) signaling pathway, followed by oxidative phosphorylation, neuroactive ligand-receptor interaction and so on. By qPCR verification, 6 genes (c6ast4, igfbp1b, mrpl42, tnnc2, emc4 and ddit4) showed consistent gene expression with those identified by transcriptome sequencing. Due to DKA-exposure, the concentrations of plasma estradiol and testosterone, and the gonado-somatic index were significantly dose-dependently declined. Also, DKA-exposure led to declining in zebrafish reproductive capacity, reflecting in fertilization, hatchability and egg production. Histopathological observations demonstrated that zebrafish ovary and testis suffered serious damage after DKA-exposure. The 4-oxo-TEMP signals increased obviously with increasing DKA-exposed concentrations, implying disruption of balance between generation and clearance of 1O2. In summary, DKAs not only produce reproductive toxicological effects on F0-zebrafish, but also result in adverse consequences for growth and development of F1-zebrafish.

Short-term toxicity assessments of an antibiotic metabolite in Wistar rats and its metabonomics analysis by ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

4-Epi-oxytetracycline (4-EOTC), one of main oxytetracycline (OTC) metabolites, can be commonly detected in food and environment. The toxicity and effects of OTC on animals have been well characterized; however, its metabolites have never been studied systemically. This study aims to investigate 15-day oral dose toxicity and urine metabonomics changes of 4-EOTC after repeated administration in Wistar rats at daily doses of 0.5, 5.0 and 50.0mg/kg bw (bodyweight). Hematology and clinical chemistry parameters, including white blood cell count, red blood cell count, total protein, globulin and albumin/globulin, were obviously altered in rats of 5.0 and 50.0mg/kg bw. Histopathology changes of kidney and liver tissues were also observed in high-dose groups. Urinary metabolites from all groups were analyzed using ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Seventeen metabolites contributing to the clusters were identified as potential biomarkers from multivariate analysis, including aminoadipic acid, 6-phosphogluconate, sebacic acid, pipecolic acid, etc. The significant changes of these biomarkers demonstrated metabonomic variations in treated rats, especially lysine and purine metabolism. For the first time in this paper, we combined the results of toxicity and metabonomics induced by 4-EOTC for the serious reconsideration of the safety and potential risks of antibiotics and its degradation metabolites.

Toxicity assessment of combined fluoroquinolone and tetracycline exposure in zebrafish (Danio rerio).

Fluoroquinolones (FQs) and tetracyclines (TCs), the two ß-diketone antibiotics (DKAs), are two frequently detected pollutants in the environment; however, little data are available on their combined toxicity to zebrafish (Danio rerio). This study reports that toxicologic effects of combined DKA (FQs-TCs) exposure on zebrafish were comparable with or slightly less than those of TCs alone, showing that TCs played a major toxicologic role in the mixtures. The effects of FQs, TCs, and DKAs on malformation rates of zebrafish were dose dependent, with EC50 values of 481.3, 16.4, and 135.1 mg/L, respectively. According to the combined effects of DKAs on zebrafish hatching, mortality, and malformation rates, the interaction between FQs and TCs was shown to be antagonistic based on three assessment methods: Toxic Unit, Additional Index, and Mixture Toxic Index. The 1.56 mg/L TC and 9.38 mg/L DKA treatments resulted in higher zebrafish basal swimming rate compared with the control group at 120 hours postfertilization (hpf). in both light and light-to-dark photoperiod experiments. Under conditions of no obvious abnormality in cardiac development, the heart beats were decreased significantly because of DKA exposure, such as decreasing by ∼20% at 150 mg/L DKAs. Transmission electron microscopy observation of myocytes from DKA-exposed hearts displayed prominent interruptions and myofibrillar disorganization of the normal parallel alignment of thick and thin filaments, and partial edematous and dissolved membranes of cell nuclear tissues. At 90 mg/L DKAs, the transcriptional levels of the acta1a, myl7, and gle1b genes, related to heart development and skeletal muscle formation, were significantly changed. This is consistent with the swimming behavior and histopathologic results obtained by transmission electron microscopy. In summary, the toxicity of the combined DKAs to zebrafish was comparable with or less than that of TCs alone and had the ability to impair individual behaviors that are of great importance in the assessment of their ecologic fitness. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 736-750, 2016.

Fluoroquinolones and Tetracycline Antibiotics in a Portuguese Aquaculture System and Aquatic Surroundings: Occurrence and Environmental Impact.

The growth of aquaculture over the past few years is widely recognized as one of the main sources of antibiotics, mainly fluoroquinolones (FQ) and tetracyclines (TC), in the aquatic environment, consequently, increasing the risk of the emergence of antibiotic bacterial resistance and promoting the spread of resistant genes. This study aimed to (1) develop and validate a multiresidue method for determination and quantification of ciprofloxacin (CIP), difloxacin (DIFL), enrofloxacin (ENR), norfloxacin (NOR), sarafloxacin (SARA), and oxytetracycline (OXY) in aquaculture waters and surrounding water bodies and (2) provide the first Portuguese data to utilize in assessment of risk of adverse effects. In addition, the potential environmental impact posed by these antibiotics to aquatic organisms, belonging to different trophic levels, when exposed to the studied aquaculture waters was also assessed. The analytical strategy comprised of solid-phase extraction (SPE) through Oasis HLB cartridges, and detection and quantification by liquid chromatography with tandem mass spectrometry (LC/MS(n)). Method detection limits (MDL) and method quantification limits (MQL) were in the range of 0.7-3 ng/L and 2.4-10 ng/L, respectively. Recoveries varied between 57.4 and 122.8%. The method was applied to 31 water samples collected from an aquaculture and surrounding water bodies located in north of Portugal. Residues of all antibiotics, except SARA and DIFL, were detected at concentrations ranging from 3 to 75.1 ng/L. Norfloxacin was the antibiotic present at highest frequency and concentration. Regarding the environmental impact assessment (EIA), a risk quotient higher than 1 was observed for NOR.

Physiological effects of tetracycline antibiotic pollutants on non-target aquatic Microcystis aeruginosa.

This study aimed to evaluate the aquatic toxicity of three typical tetracycline antibiotics, including tetracycline, oxytetracycline, and chlortetracycline, on the cyanobacterium Microcystis aeruginosa. The cell density, chlorophyll a content, protein content, and enzymatic antioxidant activities were determined. The results showed that the cell growth was significantly inhibited by the three compounds at a low concentration. The chlorophyll a and protein content decreased significantly after exposure to 0.05 mg L(-1) of each compound for 9 d. When exposed to 0.2-1 mg L(-1) of tetracycline, the superoxide dismutase (SOD) activity increased, but peroxidase (POD) and catalase (CAT) activities decreased. In contrast, when exposed to oxytetracycline and chlortetracycline at different concentrations ranging from 0.2 to 1 mg L(-1) and from 0.01 to 0.05 mg L(-1), the SOD activity decreased, but the POD and CAT activities increased. These findings indicate that tetracycline antibiotics influence cell growth and protein synthesis, and they also induce oxidative stress in M. aeruginosa at environmentally similar concentrations. Thus, this study may provide further insights into the toxic effects of tetracycline antibiotics and the controlled use of antibiotics.

Tetracyclines uptake from irrigation water by vegetables: Accumulation and antimicrobial resistance risks.

Wastewater irrigation may introduce antibiotic residues in the soil-plant systems. This study aimed to investigate the uptake of tetracyclines by spinach and collard greens and assess associated ecological and human health risks. Synthetic wastewater spiked with 1 ppm and 10 ppm of oxytetracycline, doxycycline, and tetracycline was used to grow vegetables in a greenhouse pot experiment. The uptake and accumulation of the tetracyclines were low and residual concentrations in the soil were negligible. All the tetracyclines were detected at concentrations ranging from 1.68 to 51.41 µg/g (spinach) and 1.94-30.95 µg/g (collard greens). The accumulation rate was in a dose-response scenario with a bioconcentration factor of 6.34 mL/kg (spinach) and 2.64 mL/kg (collard greens). Oxytetracycline had the highest accumulation in leaves, followed by doxycycline and tetracycline, and the residual concentrations followed the same order. The highest residual concentration was in soils receiving 10 ppm oxytetracycline. Residual concentrations in the soil were lower than accumulated levels and exerted negligible ecological risks. Tetracyclines accumulation in spinach significantly differed between the vegetables demonstrating a subspecies difference in uptake and accumulation. Ecological risk quotient (RQ) and human health risk quotient (HQ) were below thresholds that would exert toxicity and resistance selection impacts. Although RQs and HQs are low (<0.1), this study shows that the vegetables accumulate tetracyclines from irrigation water, posing plausible human health risks to allergic individuals. Similarly, the ecological risks cannot be ignored because the synergistic and antagonistic effects of sublethal concentrations can perturb ecosystem processes.

Effects of adsorption onto silica sand particles on the hydrolysis of tetracycline antibiotics.

Due to high usage of tetracycline antibiotics, concerns have been raised about their environmental fate. In this study, potential changes in the pseudo-first-order hydrolysis rate constants for three tetracyclines, tetracycline (TC), oxytetracycline (OTC) and chlortetracycline (CTC), were evaluated by measuring the rate constants in batch and column leaching experiments. The first-order hydrolysis rate constants were measured at pH 5, 7, and 9 using batch tests. The values were highest at pH 7 for all three tetracyclines (0.0030 ± 0.0004, 0.0042 ± 0.0001, and 0.0323 ± 0.0003 h(-1) for TC, OTC, and CTC, respectively), indicating relatively short environmental half-lives of tetracyclines. Interestingly, it was found that the rates of degradation of the parent tetracyclines were much faster when silica sand was present in a batch solution or when the solution was passed through a silica column. For example, the ratios of the first-order degradation rate constants obtained in the column experiments to those in batch experiments were 13.2, 2.1, and 2.0 for TC, OTC, and CTC at a volumetric flow rate of 0.08 mL h(-1), with an observed tendency for this ratio to increase with an increased flow rate. This indicates that the silica surface may serve as a catalyst for hydrolysis and that the actual environmental half-lives of tetracycline antibiotics could be shorter than those estimated from laboratory hydrolysis rate constants using the standard batch protocol. Furthermore, the toxicity of the column effluent containing hydrolysis metabolites was assessed using bioluminescence inhibition in Vibrio fischeri. It was estimated that the toxicity of the metabolites of CTC was lower than that of their parent compound, whereas the toxicity of metabolites of TC and OTC was as high as or higher than that of their parent compounds.

[Hygienic substantiation of the permissible levels for tetracycline-group antibiotics in food].

For the purpose of justification of the hygienic standard for tetracycline-group antibiotics in the food production established in the Russian Federation at more rigid level, than maximum and admissible levels (MAL) of the Codex Alimentarius Commission, the analysis of data of literature on negative nature of impact of low concentration of these antibiotics on an organism and the environmental conditions and risk for health has been performed. Inadequacy of the accepted admissible daily dose (ADD) accepted by The Joint FAO/WHO Expert Committee on Food Additives (JECFA) on action on selection of resistant E. coli in intestines, for the wide contingent of consumers in connection with ignoring of obvious factors of uncertainty (gastrointestinal dysbiosis, age and individual variations in the microbiota of people synergy with other antibiotics residues in food and indirect impact on an organism through microflora from the natural habitat (resistance genes, modified causative organisms with altered properties).. By the analysis of information received with the use of modern molecular and genetic methods, the role of Subinhibitory concentrations (sub-MICs) of tetracyclines as biologically active substances, signaling molecules which, without causing obvious negative consequences in a macroorganism, serve as a major factor of regulation of a transcription in microorganisms and activation of a horizontal gene transfer coding resistance, transferred on conjugative transposons of Tn916-Tn1545 family. Reasonable scientific data on a dominating contribution of minor levels of tetracyclines in globalization in the nature of the most adverse transmissive type of the antibiotic resistance interfaced to formation new bacterial pathotypes, as consequences of irrationally high scales of application in agriculture and strengthened impact on microbic ecosystems of live organisms and objects of habitat are presented. For minimization of this mediated risk for health the need of preservation of operating level of the tetracyclines residues (by < or = 0,01 mg/kg of a product), MAL which were unlike Codex MAL (< or = 0,1-1,2 mg/kg) in a zone of concentrations below 0,1 Misc not capable to initiation of the above described changes has been proved, till up to receipt of new scientific data on influence on macro - or microorganisms of the doses equal or below this value on macro - or microorganisms.

Binding of the highly toxic tetracycline derivative, anhydrotetracycline, to bovine serum albumin.

Tetracycline (TC) derivatives are extensively used as antibiotics in human and animal medicine and, very recently, they have been screened as anti-amyloidogenic drugs. Anhydrotetracycline (AHTC) is one of the major degradation products of TC that has been linked to several side effects of the drug. We evaluated the interaction of AHTC with bovine serum albumin (BSA), one of the main carriers of amphiphilic molecules in blood, using three complementary analytical methods: fluorescence spectroscopy, isothermal titration calorimetry and differential scanning calorimetry. AHTC bound to BSA with an association constant in the order of 10(5) M(-1). Drug binding was enthalpically and entropically driven and seemed to involve hydrophobic interactions. AHTC fluorescence enhancement and hypsochromic shifts observed upon binding suggested a low-polarity location excluded from water for the bound drug. Our data are useful for evaluating the biodisponibility of the pharmacophore and the dynamic distribution of the toxic derivative.

Tetracyclines in the environment: An overview on the occurrence, fate, toxicity, detection, removal methods, and sludge management.

Tetracyclines (TCs) are a group of broad-spectrum antibiotics having vast human, veterinary, and aquaculture applications. The continuous release of TCs residues into the environment and the inadequate removal through the conventional treatment systems result in its prevalent occurrence in soil, surface water, groundwater, and even in drinking water. As aqueous TCs contamination is the tip of the iceberg, and TCs possess good sorption capacity towards soil, sediments, sludge, and manure, it is insufficient to rely on the sorptive removal in the conventional water treatment plants. The severity of the TCs contamination is evident from the emergence of TCs resistance in a wide variety of microorganisms. This paper reviews the recent research on the TCs occurrence in the environmental matrices, fate in natural systems, toxic effects, and the removal methods. The high performance liquid chromatography (HPLC) determination of TCs in environmental samples and the associated technology developments are analyzed. The benefits and limitations of biochemical and physicochemical removal processes are also discussed. This work draws attention to the inevitability of proper TC sludge management. This paper also gives insight into the limitations of TCs related research and the future scope of research in environmental contamination by TCs residues.

Occurrence, fate, and risk assessment of typical tetracycline antibiotics in the aquatic environment: A review.

Tetracyclines (TCs), used as human and veterinary medicines, are the most widely used antibiotics. More than 75% of TCs are excreted in an active form and released into the environment through human and animal urine and feces, causing adverse effects on the ecological system and human health. Few articles review the environmental occurrence and behaviors of TCs, as well as their risks and toxicities. Here, we comprehensively summarized the recent advances on the following important issues: (1) Environmental occurrence of TCs. TCs are used globally and their occurrence in the aquatic environment has been documented, including surface water, groundwater, drinking water, wastewater, sediment, and sludge. (2) Environmental behaviors of TCs, particularly the fate of TCs in wastewater treatment plants (WWTPs). Most WWTPs cannot effectively remove TCs from wastewater, so alternative methods for efficient removal of TCs need to be developed. The latest degradation methods of TCs are summarized, including adsorption, photocatalytic, photochemical and electrochemical, and biological degradations. (3) Toxicities and possible risks of TCs. The toxicological data of TCs indicate that several TCs are more toxic to algae than fish and daphnia. Risk assessments based on individual compound exposure indicate that the risks arising from the current concentrations of TCs in the aquatic environment cannot be ignored.

Application of microbial assay for risk assessment biotest in evaluation of toxicity of human and veterinary antibiotics.

The microbial assay for risk assessment (MARA) is a multispecies, growth inhibition microplate toxicity test with 11 microorganisms individually lyophilized in microplate wells. The microbial species representing wide diversity, generated 11 microbial toxic concentration (MTC) values were obtained giving a unique "toxic fingerprint" profile of the test sample. The toxicity of 14 antibiotics was evaluated with the MARA test. The fingerprints for each group of antibiotics differ, indicating a particular toxic profile. Tetracyclines were the most toxic antibacterials with the minimum MTC value of 3.6 µg L(-1). In the group of tetracyclines the order of the three most sensitive microbial strains was the same 2 > 6 > 7. Quinolones affected the most sensitive strain(s) at concentrations of 12-75 µg L(-1). The MARA bioassay exhibited different spectra of toxic responses to microbial strains for the first and second generation quinolones. However, for first generation quinolones strain 6 was substantially more sensitive than the other microorganisms, while second generation quinolones were most toxic to strain 3, followed by 6 then 4. In this instance, the three strains belong to two different phylogenetic groups-strain 3 is γ-proteotype and strains 4 and 6 are ß-proteotype.

Effects of divalent copper on microbial community, enzymatic activity and functional genes associated with nitrification and denitrification at tetracycline stress.

The effects of divalent copper (Cu(II)) on microbial community, enzymatic activity and functional genes in a sequencing batch reactor (SBR) at tetracycline (TC) stress were investigated. The enzymatic activity and functional genes abundance associated with nitrification and denitrification at a 20 mg L-1 TC stress were higher than those at a mixtures stress of 20 mg L-1 TC and 10 mg L-1 Cu(II), while they were lower than those at a mixtures stress of 20 mg L-1 TC and 40 mg L-1 Cu(II). Compared to lactate dehydrogenase (LDH) release and reactive oxygen species (ROS) production at a 20 mg L-1 TC stress, they were lower at the TC stress with 10 mg L-1 Cu(II), while they were higher at the TC stress with 40 mg L-1 Cu(II). The incremental Cu(II) concentration at a 20 mg L-1 TC stress could not change the result that the sensitivity of denitrifying enzymatic activity to TC was higher than nitrifying enzymatic activity. Compared to the relative abundance of nitrifers and denitrifers at a 20 mg L-1 TC stress, the 10 mg L-1 Cu(II) addition resulted in their increase, while they decreased as the 40 mg L-1 Cu(II) addition. The relative abundance of genera Pseudomonas, Rivibacter and Nitrobacter at the stress of Cu(II) and TC were higher than those at TC stress, suggested they had an ability to resist the stress of Cu(II) and TC.

Deciphering the microbial and genetic responses of anammox biogranules to the single and joint stress of zinc and tetracycline.

The feasibility of using anaerobic ammonium oxidation (anammox) process to treat wastewaters containing antibiotics and heavy metals was evaluated in this study. The nitrogen removal performance and characteristic parameters were monitored during the whole experimental period of 258 d. The single and joint effects of zinc and tetracycline on the microbial community were studied in upflow anaerobic sludge blanket (UASB) reactors. The anammox performance remained at levels comparable with the initial state at the lower inhibitor concentrations (zinc, 0-2.26 mg L-1; tetracycline, 0-0.5 mg L-1). When the concentrations of zinc and tetracycline increased to 3.39 mg L-1 in R1 and 1.0 mg L-1 in R2, an obvious deterioration in performance was observed. Dual inhibitors with a total concentration of ≥3 mg L-1 caused dramatic decreases in the nitrogen removal efficiency of R3. The quantification results showed that the abundances of eight antibiotic resistance genes (ARGs), czcA and intI1 in the experimental reactors generally increased under stress from metals or/and antibiotics, with final values higher than in the control, while the functional gene abundances were lower. Moreover, most genes exhibited significant correlations. Microbial community analysis indicated that Planctomycetes (represented by Candidatus Kuenenia) was inhibited by both zinc and tetracycline, but still held the dominant position. Furthermore, Caldilinea (belonging to Chloroflexi) maintained a higher abundance during the inhibitory period, implying its potential resistance to both inhibitors. These findings suggested that anammox could be inhibited by metals and antibiotics, but it has the potential to remove nitrogen from wastewaters containing both of them within the concentration threshold.