Oxytetracycline Increases the Residual Risk of Imidacloprid in Radish (Raphanus sativus) and Disturbs the Plant-Rhizosphere Microbiome Holobiont Homeostasis.

Antibiotics can be accidentally introduced into farmland by wastewater irrigation, and the environmental effects are still unclear. In this study, the effects of oxytetracycline on the residue of imidacloprid in soil and radishes were investigated. Besides, the rhizosphere microbiome and radish metabolome were analyzed. It showed that the persistence of imidacloprid in soil was unchanged, but the content of olefin-imidacloprid was increased by oxytetracycline. The residue of imidacloprid in radishes was increased by nearly 1.5 times, and the hazard index of imidacloprid was significantly raised by 1.5-4 times. Oxytetracycline remodeled the rhizosphere microbiome, including Actinobe, Elusimic, and Firmicutes, and influenced the metabolome of radishes. Especially, some amino acid metabolic pathways in radish were downregulated, which might be involved in imidacloprid degradation. It can be assumed that oxytetracycline increased the imidacloprid residue in radish through disturbing the plant-rhizosphere microbiome holobiont and, thus, increased the pesticide dietary risk.

miRNA-seq analysis of liver tissue from largemouth bass (Micropterus salmoides) in response to oxytetracycline and enzyme-treated soy protein.

The specific miRNA regulation triggered by enzyme-treated soybean protein in response to well-known stressors, such as the prophylactic use of the antimicrobial oxytetracycline, remains unknown. Hence, this study aimed to evaluate the regulatory changes of hepatic miRNAs induced by oxytetracycline and enzyme-treated soybean protein in largemouth bass dietary formulations. The experiment was designed with three groups: the normal control (NC), the oxytetracycline exposure treatment group (OTC), and the pre-treatment with enzyme-treated soybean protein before oxytetracycline exposure group (ETSP). miRNA sequencing was employed to characterize the differences between these groups. In conclusion, the NC group exhibited up-regulation of 13 host miRNAs and down-regulation of 1 miRNA compared to the OTC group, whereas the ETSP group showed an increasing trend of 36 host miRNAs and a decreasing trend of 13 host miRNAs compared to the OTC group. Nine miRNAs were identified as prudential targets for enzyme-treated soy protein, protecting the largemouth bass liver from oxytetracycline. Furthermore, gene ontology analysis revealed nine key miRNAs that mediate signaling pathways with significant differences. The cellular lipid metabolic process was identified as the most important biological process, and the propanoate metabolism pathway was highlighted as significant. These results will facilitate further exploration of the mechanism by which enzyme-treated soy protein alleviates the effects of oxytetracycline on largemouth bass in water environments.

Potential mechanism of biochar enhanced degradation of oxytetracycline by Pseudomonas aeruginosa OTC-T.

Extensive use of oxytetracycline (OTC) and the generation of its corresponding resistance genes have resulted in serious environmental problems. Physical-biological combined remediation is an attractive method for OTC degradation because of its high remediation efficiency, stability, and environmental friendliness. In this study, an effective OTC-degrading strain identified as Pseudomonas aeruginosa OTC-T, was isolated from chicken manure. In the degradation experiment, the degradation rates of OTC in the degradation systems with and without the biochar addition were 92.71-100 % and 69.11-99.59 %, respectively. Biochar improved the tolerance of the strain to extreme environments, and the OTC degradation rate increased by 20.25 %, 18.61 %, and 13.13 % under extreme pH, temperature, and substrate concentration conditions, respectively. Additionally, the degradation kinetics showed that biochar increased the reaction rate constant in the degradation system and shortened the degradation period. In the biological toxicity assessment, biochar increased the proportion of live cells by 17.63 % and decreased the proportion of apoptotic cells by 58.87 %. Metabolomics revealed that biochar had a significant effect on the metabolism of the strains and promoted cell growth and reproduction, effectively reducing oxidative stress induced by OTC. This study elucidates how biochar affects OTC biodegradation and provides insights into the future application of biochar-assisted microbial technology in environmental remediation.

Silymarin enhances the response to oxytetracycline treatment in Oreochromis niloticus experimentally infected with Aeromonas hydrophila.

Many governments have approved the use of oxytetracycline as an antibiotic additive to food fish, with oxytetracycline now routinely used in many nations. However, oxytetracycline is known to have immunosuppression impacts. We, therefore, evaluated the immunological, antioxidative, and histopathological status of Nile tilapia fed a diet containing silymarin (100 mg/kg fish feed) for 0, 2, 4, 6, and 8 weeks. The protective effects of silymarin against Aeromonas hydrophila (A. hydrophila) infection and oxytetracycline treatment were evaluated. Blood parameters (erythrocyte count, white blood cell count, hemoglobin, and packed cell volume) improved over time in fish fed on dietary silymarin. Serum levels of alanine aminotransferase (ALT) were lower in fish fed on dietary silymarin, whereas serum levels of aspartate transferase (AST)and alkaline phosphatase (ALK) were unchanged. Dietary silymarin affected serum lipid profiles as decreases in serum triglyceride and low-density lipoprotein cholesterol levels and a trend toward lower cholesterol levels, whereas serum high-density lipoprotein cholesterol levels were increased compared to fish fed on the control diet. Dietary silymarin resulted in an increase of serum total protein levels and globulin fractions. Significant and progressive increases in catalase and glutathione peroxidase levels were observed after six weeks of feeding on a dietary silymarin before decreasing to control levels at the end of the experimental period. Fish fed on dietary silymarin, interleukin-1 and fish tumor necrosis factor-alpha were upregulated in hepatic tissues; however, interleukin-10 levels decreased to comparable levels to controls after eight weeks. Fish infected with A. hydrophila displayed septicemia (opaque eye, hemorrhagic ulcers, dentated fins, hepatomegaly, and splenomegaly). Reduced mortality was observed in Nile tilapia infected with A. hydrophila and fed a diet containing silymarin, indicating that silymarin improves fish responses to oxytetracycline with a 37% reduction in mortality.

Biochar-assisted degradation of oxytetracycline by Achromobacter denitrificans and underlying mechanisms.

Contamination of the environment with large amounts of residual oxytetracycline (OTC) and the corresponding resistance genes poses a potential threat to natural ecosystems and human health. In this study, an effective OTC-degrading strain, identified as Achromobacter denitrificans OTC-F, was isolated from activated sludge. In the degradation experiment, the degradation rates of OTC in the degradation systems with and without biochar addition were 95.01-100% and 73.72-99.66%, respectively. Biochar promotes the biodegradation of OTC, particularly under extreme environmental conditions. Toxicity evaluation experiments showed that biochar reduced biotoxicity and increased the proportion of living cells by 17.36%. Additionally, biochar increased the activity of antioxidant enzymes by 34.1-91.0%. Metabolomic analysis revealed that biochar promoted the secretion of antioxidant substances such as glutathione and tetrahydrofolate, which effectively reduced oxidative stress induced by OTC. This study revealed the mechanism at the molecular level and provided new strategies for the bioremediation of OTC in the environment.

Tetracycline-modifying enzyme SmTetX from Stenotrophomonas maltophilia.

The resistance of the emerging human pathogen Stenotrophomonas maltophilia to tetracycline antibiotics mainly depends on multidrug efflux pumps and ribosomal protection enzymes. However, the genomes of several strains of this Gram-negative bacterium code for a FAD-dependent monooxygenase (SmTetX) homologous to tetracycline destructases. This protein was recombinantly produced and its structure and function were investigated. Activity assays using SmTetX showed its ability to modify oxytetracycline with a catalytic rate comparable to those of other destructases. SmTetX shares its fold with the tetracycline destructase TetX from Bacteroides thetaiotaomicron; however, its active site possesses an aromatic region that is unique in this enzyme family. A docking study confirmed tetracycline and its analogues to be the preferred binders amongst various classes of antibiotics.

Stress of cupric ion and oxytetracycline in Chlorella vulgaris cultured in swine wastewater.

Chlorella culturing has the advantages in treatment of wastewater including swine wastewater from anaerobic digesters due to the product of biolipids and the uptake of carbon dioxide. However, there often exist high concentrations of antibiotics and heavy metals in swine wastewater which could be toxic to chlorella and harmful to the biological systems. This study examined the stress of cupric ion and oxytetracycline (OTC) at various concentrations on the nutrient removal and biomass growth in Chlorella vulgaris culturing in swine wastewater from anaerobic digesters, and its biochemical responses were also studied. Results showed that dynamic hormesis of either OTC concentration or cupric ion one on Chlorella vulgaris were confirmed separately, and the presence of OTC not only did not limit biomass growth and lipids content of Chlorella vulgaris but also could mitigate the toxicity of cupric ion on Chlorella vulgaris in combined stress of Cu2+ and OTC. Extracellular polymeric substances (EPS) of Chlorella vulgaris were used to explain the mechanisms of stress for the first time. The content of proteins and carbohydrates in EPS increased, and the fluorescence spectrum intensity of tightly-bound EPS (TB-EPS) of Chlorella vulgaris decreased with increasing concentration of stress because Cu2+ and OTC may be chelated with proteins of TB-EPS to form non-fluorescent characteristic chelates. The low concentration of Cu2+ (≤1.0 mg/L) could enhance the protein content and promote the activity of superoxide dismutase (SOD) while these parameters were decreased drastically under 2.0 mg/L of Cu2+. The activity of adenosine triphosphatase (ATPase) and glutathione (GSH) enhanced with the increase of OTC concentration under combined stress. This study helps to comprehend the impact mechanisms of stress on Chlorella vulgaris and provides a novel strategy to improve the stability of microalgae systems for wastewater treatment.

Evidence for the feasibility of transmembrane proton gradient regulating oxytetracycline extracellular biodegradation mediated by biosynthesized palladium nanoparticles.

Extracellular biodegradation is a promising technology for removing antibiotics and repressing the spread of resistance genes, but the strategy is limited by the low extracellular electron transfer (EET) efficiency of microorganisms. In this work, biogenic Pd0 nanoparticles (bio-Pd0) were introduced in cells in situ to enhance oxytetracycline (OTC) extracellular degradation and the effects of transmembrane proton gradient (TPG) on EET and energy metabolism mediated by bio-Pd0 were investigated. The results indicated that the intracellular OTC concentration gradually decreased with increase in pH due to the simultaneous decreases of OTC adsorption and TPG-dependent OTC uptake. On the contrary, the efficiency of OTC biodegradation mediated by bio-Pd0@B. megaterium showed a pH-dependent increase. The negligible intracellular OTC degradation, the high dependence of OTC biodegradation on respiration chain and the results on enzyme activity and respiratory chain inhibition experiments showed that NADH-dependent (rather than FADH2-dependent) EET process mediated by substrate-level phosphorylation modulated OTC biodegradation due to high energy storage and proton translocation capacity. Moreover, the results showed that altering TPG is an efficient approach to improve EET efficiency, which can be attributed to the increased NADH generation by the TCA cycle, enhanced transmembrane electron output efficiency (as evidenced by increased intracellular electron transfer system (IETS) activity, the negative shift of onset potential, and enhanced one-electron transfer through bound flavin) and stimulation of substrate-level phosphorylation energy metabolism catalyzed by succinic thiokinase (STH) under low TPG conditions. The results of structural equation model that OTC biodegradation was directly and positively modulated by the net outward proton flux as well as STH activity, and indirectly regulated by TPG through NADH level and IETS activity confirmed the previous findings. This study provides a new perspective for engineering microbial EET and application of bioelectrochemistry processes in bioremediation.

Assessment of Nonalcoholic Fatty Liver Disease Symptoms and Gut-Liver Axis Status in Zebrafish after Exposure to Polystyrene Microplastics and Oxytetracycline, Alone and in Combination.

BACKGROUND: Environmental pollution may give rise to the incidence and progression of nonalcoholic fatty liver disease (NAFLD), the most common cause for chronic severe liver lesions. Although knowledge of NAFLD pathogenesis is particularly important for the development of effective prevention, the relationship between NAFLD occurrence and exposure to emerging pollutants, such as microplastics (MPs) and antibiotic residues, awaits assessment. OBJECTIVES: This study aimed to evaluate the toxicity of MPs and antibiotic residues related to NAFLD occurrence using the zebrafish model species. METHODS: Taking common polystyrene MPs and oxytetracycline (OTC) as representatives, typical NAFLD symptoms, including lipid accumulation, liver inflammation, and hepatic oxidative stress, were screened after 28-d exposure to environmentally realistic concentrations of MPs (0.69mg/L) and antibiotic residue (3.00µg/L). The impacts of MPs and OTC on gut health, the gut-liver axis, and hepatic lipid metabolism were also investigated to reveal potential affecting mechanisms underpinning the NAFLD symptoms observed. RESULTS: Compared with the control fish, zebrafish exposed to MPs and OTC exhibited significantly higher levels of lipid accumulation, triglycerides, and cholesterol contents, as well as inflammation, in conjunction with oxidative stress in their livers. In addition, a markedly smaller proportion of Proteobacteria and higher ratios of Firmicutes/Bacteroidetes were detected by microbiome analysis of gut contents in treated samples. After the exposures, the zebrafish also experienced intestinal oxidative injury and yielded significantly fewer numbers of goblet cells. Markedly higher levels of the intestinal bacteria-sourced endotoxin lipopolysaccharide (LPS) were also detected in serum. Animals treated with MPs and OTC exhibited higher expression levels of LPS binding receptor (LBP) and downstream inflammation-related genes while also exhibiting lower activity and gene expression of lipase. Furthermore, MP-OTC coexposure generally exerted more severe effects compared with single MP or OTC exposure. DISCUSSION: Our results suggested that exposure to MPs and OTC may disrupt the gut-liver axis and be associated with NAFLD occurrence. https://doi.org/10.1289/EHP11600.

Efficiency of conventional and nanoparticle oxytetracycline in treatment of clinical endometritis in postpartum dairy cows.

The incidence of clinical endometritis in dairy cows postpartum is one of the important reasons for financial losses in the dairy industry. The costs of treatment, milk losses, infertility, repeated breeding, and high annual culling rate of dairy cows present immediate losses in case of treatment failure. The commonly used therapeutic methods for clinical endometritis have not been successful nor have given definitive solutions to overcome the complications of the disease in dairy cows. Therefore, it was necessary to propose an innovative treatment program to overcome the reasons for the failure and lack of effectiveness of the treatment of clinical endometritis. This was tackled in the current study; oxytetracycline with different concentrations, oxytetracycline 5% (OTCC5%), oxytetracycline 20% (OTCC20%), and oxytetracycline 20% nanoparticles (OTC-NPs) were used for the treatment of clinical endometritis. Diagnosis of clinical endometritis was based on the assessment of high serum concentration of pro-inflammatory cytokines, acute phase protein, increased endometrium thickness, and intrauterine discharges with different degrees of echogenicity monitored by ultrasonography. Application of OTC-NPs revealed a decrease in serum concentration of pro-inflammatory cytokines (IL-1, IL-6, and TNF-α) and acute phase proteins compared to OTCC20% and OTCC5% groups. The improvement achieved by OTC-NPs may be attributed to the reduction of OTC particles into nano size which facilitates its tissue bioavailability, dispersion, penetration power to deeper tissues, and its more broad-spectrum activities. These activities were clearly apparent after the evacuation of uterine contents using a single dose of PGF2α. The OTC-NPs revealed a reduction in serum concentration of cytokines compared to OTCC20% and OTCC5% groups arranged as follows: 10.11, 25.45, 35.56 for IL-1; 99, 300, 319 for IL-6; 1.01, 4.40, 8.06 for CRP; and 46, 183, 266 for TNF-α. Furthermore, an increase in serum concentration of albumin (3.34) was obtained by OTC-NPs compared to OTCC5% (1.70). This improvement can be taken as evidence of liver resumption functions and inflammatory reactions. On the other side, globulin concentration recorded an increase like albumin and total proteins in OTC-NPs compared to others. A reduction in the endometrium thickness in OTC-NPs with the disappearance of intrauterine discharges was monitored by ultrasonography. This confirmed the subsiding of clinical endometritis in OTC-NPs group. Moreover, a significant improvement in conception and pregnancy rate in OTC-NPs compared to other groups were observed.

Effects of antibiotics stress on root development, seedling growth, antioxidant status and abscisic acid level in wheat (Triticum aestivum L.).

The veterinary antibiotics contamination in agroecosystems is a substantial problem globally. However, little is known about their toxicity to crops, especially in wheat. This study evaluated the phytotoxic effects of the two most representative antibiotics, namely oxytetracycline (OTC) and enrofloxacin (ENR), on seed germination, seedling growth, root elongation and antioxidant status in wheat, and investigated the response of abscisic acid (ABA) to antibiotic stress and its underlying mechanism. The results showed that OTC and ENR under the experimental concentrations (5, 10, 20, 40 and 80 mg·L-1) had no influence on seed germination of wheat. The reduced root length, fresh weight and surface area were observed when the concentrations of OTC and ENR were higher than 10 mg·L-1 and 5 mg·L-1, respectively. High concentrations (＞40 mg·L-1) of antibiotics dramatically decreased the root length, fresh weight, root numbers and surface area as well as the number of stele cells and stele area. The activity of catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD), and malondialdehyde (MDA) content in shoots and roots were increased with the increasing OTC and ENR concentrations. High concentrations (＞40 mg·L-1) of antibiotics improved ABA content and enhanced the transcription levels of genes related to ABA biosynthesis (TaNCED1 and TaNCED2) and metabolism (TaABA8'OH1-A and TaABA8'OH2-A) in shoots and roots of wheat seedlings. Wheat seedlings had relatively strong sensitivity to low concentration (5 mg·L-1) of ENR. These results suggest that OTC and ENR modulate root development and seedling growth by regulating ABA level and antioxidant defense system in wheat.

Antibiotics induced changes in nitrogen metabolism and antioxidative enzymes in mung bean (Vigna radiata).

Excessive use and release of antibiotics into the soil environment in the developing world have resulted in altered soil processes affecting terrestrial organisms and posing a serious threat to crop growth and productivity. The present study investigated the influence of exogenously applied oxytetracycline (OXY) and levofloxacin (LEV) on plant physiological responses, key enzymes involved in nitrogen metabolism (e.g., nitrate reductase, glutamine synthetase), nitrogen contents and oxidative stress response of mung bean (Vigna radiata). Plants were irrigated weekly with antibiotics containing water for exposing the plants to different concentrations i.e., 1, 10, 20, 50, and 100 mg L-1. Results showed a significant decrease in nitrate reductase activity in both antibiotic treatments and their mixtures and increased antioxidant enzymatic activities in plants. At lower concentrations of antibiotics (≤20 mg L-1), 53.9 % to 78.4 % increase in nitrogen content was observed in levofloxacin and mixtures compared to the control, resulting in an increase in the overall plant biomass. Higher antibiotic (≥50 mg L-1) concentration showed 58 % decrease in plant biomass content and an overall decrease in plant nitrogen content upon exposure to the mixtures. This was further complemented by 22 % to 42 % increase in glutamine synthetase activity observed in the plants treated with levofloxacin and mixtures. The application of low doses of antibiotics throughout the experiments resulted in lower toxicity symptoms in the plants. However, significantly higher malondialdehyde (MDA) concentrations at higher doses (20 mg L-1 and above) than the control showed that plants' tolerance against oxidative stress was conceded with increasing antibiotic concentrations. The toxicity trend was: levofloxacin > mixture > oxytetracycline.

Removal performance and inhibitory effects of combined tetracycline, oxytetracycline, sulfadiazine, and norfloxacin on anaerobic digestion process treating swine manure.

Combined veterinary antibiotics (CVAs) belonging to different antibiotics classes could cause exacerbated impacts on the anaerobic digestion (AD) process of swine manure. Four different antibiotics "two tetracyclines: tetracycline (TC) and oxytetracycline (OTC), one fluoroquinolones: norfloxacin (Norf), and one sulfonamides: sulfadiazine (SDZ)" were combined to evaluate their removal performances and its inhibition effects on AD. Results indicated that CVAs removal decreased from 84.3 to 63.7 %, with an increase in the initial concentration from 12.5 to 50 mg L-1, where the removal of CVAs occurring in the order OTC > TC > Norf > SDZ. An average of 9.5, 7.5, 9.5, and 32.1 % of the spiked TC, OTC, SDZ, and Norf were remained in the sludge, respectively. With 50 mg L-1 of CVAs, a competitive adsorption phenomenon was found to have a notable impact on biodegradation microorganisms' activity leading a 73.1 % decrease in CH4 production. CVAs caused a temporal inhibition to the acidogenic activity followed by partial inhibition to methanogenic by 66.8 %, and IC50 was 38.5 mg L-1. Moreover, CVAs resulted in acetate accumulation, while 26 % and 48 % lower in TS and COD removal, respectively, were observed. A significant reduction in the relative abundance of bacteria and archaeal genera was also mentioned. The findings of this research would provide a more in-depth understanding of AD's performance in treating swine manure contaminated with combined antibiotics.

Antibacterial properties and in silico modeling perspective of nano ZnO transported oxytetracycline-Zn2+ complex [ZnOTc]+ against oxytetracycline-resistant Aeromonas hydrophila.

Emergence of antibiotics resistance has threatening consequences not only for human health but also for animal health issues in agriculture. Several animal pathogenic bacteria have developed antibiotic resistance and managing same has tremendous cost repercussions and may lead to total harvest loss. Hence in the present study, efforts are made to revitalize an old antibiotic molecule, oxytetracycline (OTc), through nanodelivery approaches using zinc oxide nanoparticles (nZnO) to confront OTc resistant fish pathogenic bacteria Aeromonas hydrophila. OTc was impregnated in nZnO through in situ precipitation method to develop OTc loaded ZnO nanoparticles (OTc@nZnO) with average size of 99.42 nm. Spectroscopic investigation of same revealed complexation of Zn2+ with amide and aromatic carbonyl moieties of OTc [ZnOTc]+. The complex performed better against A. hydrophila with 7-15 mm inhibition zone as compared to nil for bare OTc at same dose. OTc also showed MIC of 150 µg ml-1 and for OTc@nZnO it was 7.02 µg ml-1 with faster killing rate (k, -0.95). In silico docking simulation suggest that [ZnOTc]+ had low binding affinity (LBE > -5.00 kcal mol-1) toward TetR(E) and TetA(E) proteins of A. hydrophila as compared to OTc (LBE < -8.00 kcal mol-1). This study postulates that [ZnOTc]+ released from OTc@nZnO can escape TetR(E) and TetA(E) resistance proteins and bind at 30S ribosomal subunit with high affinity (<-11.00 kcal mol-1) to exert antibacterial properties. In the recent scenario of recurrent antimicrobial resistance, the develop antibiotic-nanocomposites could come out as potential solution, however further study is required for its feasibility for use in animal health care.

Removal of oxytetracycline and ofloxacin in wastewater by microalgae-bacteria symbiosis for bioenergy production.

The development of microalgae-bacteria symbiosis for treating wastewater is flourishing owing to its high biomass productivity and exceptional ability to purify contaminants. A nature-selected microalgae-bacteria symbiosis, mainly consisting of Dictyosphaerium and Pseudomonas, was used to treat oxytetracycline (OTC), ofloxacin (OFLX), and antibiotic-containing swine wastewater. Increased antibiotic concentration gradually reduced biomass productivity and intricately changed symbiosis composition, while 1 mg/L OTC accelerated the growth of symbiosis. The symbiosis biomass productivity reached 3.4-3.5 g/L (5.7-15.3 % protein, 18.4-39.3 % carbohydrate, and 2.1-3.9 % chlorophyll) when cultured in antibiotic-containing swine wastewater. The symbiosis displayed an excellent capacity to remove 76.3-83.4 % chemical oxygen demand, 53.5-62.4 % total ammonia nitrogen, 97.5-100.0 % total phosphorus, 96.3-100.0 % OTC, and 32.8-60.1 % OFLX in swine wastewater. The microbial community analysis revealed that the existence of OTC/OFLX increased the richness and evenness of microalgae but reduced bacteria species in microalgae-bacteria, and the toxicity of OFLX to bacteria was stronger than that of OTC.

Momomycin, an Antiproliferative Cryptic Metabolite from the Oxytetracycline Producer Streptomyces rimosus.

Natural products provide an important source of pharmaceuticals and chemical tools. Traditionally, assessment of unexplored microbial phyla has led to new natural products. However, with every new microbe, the number of orphan biosynthetic gene clusters (BGC) grows. As such, the more difficult proposition is finding new molecules from well-studied strains. Herein, we targeted Streptomyces rimosus, the widely-used oxytetracycline producer, for the discovery of new natural products. Using MALDI-MS-guided high-throughput elicitor screening (HiTES), we mapped the global secondary metabolome of S. rimosus and structurally characterized products of three cryptic BGCs, including momomycin, an unusual cyclic peptide natural product with backbone modifications and several non-canonical amino acids. We elucidated important aspects of its biosynthesis and evaluated its bioactivity. Our studies showcase HiTES as an effective approach for unearthing new chemical matter from "drained" strains.

Quantitative Proteomics Reveals That the Protein Components of Outer Membrane Vesicles (OMVs) in Aeromonas hydrophila Play Protective Roles in Antibiotic Resistance.

In recent years, the intracellular mechanisms that contribute to antibiotic resistance have received increasing attention, and outer membrane vesicles (OMVs) have been reported to be related to antibiotic resistance in several Gram-negative bacterial species. However, the intrinsic molecular mechanisms and the form of such antibiotic resistance are still largely unknown. In this study, OMVs from an oxytetracycline (OXY) sensitive aquatic pathogen, Aeromonas hydrophila (OXY-S), were found with significantly increased OXY resistance. Interestingly, the OXY-resistant strain (OXY-R) had a more protective role in OXY resistance. Therefore, a DIA-based quantitative proteomics analysis was performed to compare the differential expression of OMV proteins between OXY-R (OMVsR) and OXY-S (OMVsS). The results showed that seven proteins increased and five proteins decreased in OMVsR vs OMVsS. A subsequent antibiotics susceptibility assay showed that the deletion of icd, rpsF, and iscS significantly increased OXY sensitivity. Moreover, the exogenous addition of the crude OMV fractions of overexpressed recombinant proteins in E. coli with rRpsF, rIcd, rIscS, rOmpA, rPepA, rFrdA, and rRplQ demonstrated that these proteins promoted the OXY resistance of A. hydrophila. Overall, our results indicate the important protective role of OMVs in antibiotic resistance in A. hydrophila and provide novel insights on bacterial antibiotic resistance mechanisms.

The Lysine Acetylation Modification in the Porin Aha1 of Aeromonas hydrophila Regulates the Uptake of Multidrug Antibiotics.

Protein lysine acetylation (Kac) modification plays important roles in diverse physiological functions. However, there is little evidence on the role of Kac modification in bacterial antibiotic resistance. Here, we compared the differential expressions of whole-cell proteins and Kac peptides in oxytetracycline sensitive and oxytetracycline resistance (OXYR) strains of Aeromonas hydrophila using quantitative proteomics technologies. We observed a porin family protein Aha1 downregulated in the OXYR strain, which may have an important role in the OXY resistance. Interestingly, seven of eight Kac peptides of Aha1 decreased abundance in OXYR as well. Microbiologic assays showed that the K57R, K187R, and K197R Aha1 mutants significantly increased antibiotic resistance to OXY and reduced the intracellular OXY accumulation in OXY stress. Moreover, these Aha1 mutants displayed multidrug resistance features to tetracyclines and ß-lactam antibiotics. The 3D model prediction showed that the Kac states of K57, K187, and K197 sites located at the extracellular pore vestibule of Aha1 may be involved in the uptake of specific types of antibiotics. Overall, our results indicate a novel antibiotic resistance mechanism mediated by Kac modification, which may provide a clue for the development of antibiotic therapy strategies.

Reference-Grade Genome and Large Linear Plasmid of Streptomyces rimosus: Pushing the Limits of Nanopore Sequencing.

Streptomyces rimosus ATCC 10970 is the parental strain of industrial strains used for the commercial production of the important antibiotic oxytetracycline. As an actinobacterium with a large linear chromosome containing numerous long repeat regions, high GC content, and a single giant linear plasmid (GLP), these genomes are challenging to assemble. Here, we apply a hybrid sequencing approach relying on the combination of short- and long-read next-generation sequencing platforms and whole-genome restriction analysis by using pulsed-field gel electrophoresis (PFGE) to produce a high-quality reference genome for this biotechnologically important bacterium. By using PFGE to separate and isolate plasmid DNA from chromosomal DNA, we successfully sequenced the GLP using Nanopore data alone. Using this approach, we compared the sequence of GLP in the parent strain ATCC 10970 with those found in two semi-industrial progenitor strains, R6-500 and M4018. Sequencing of the GLP of these three S. rimosus strains shed light on several rearrangements accompanied by transposase genes, suggesting that transposases play an important role in plasmid and genome plasticity in S. rimosus. The polished annotation of secondary metabolite biosynthetic pathways compared to metabolite analysis in the ATCC 10970 strain also refined our knowledge of the secondary metabolite arsenal of these strains. The proposed methodology is highly applicable to a variety of sequencing projects, as evidenced by the reliable assemblies obtained. IMPORTANCE The genomes of Streptomyces species are difficult to assemble due to long repeats, extrachromosomal elements (giant linear plasmids [GLPs]), rearrangements, and high GC content. To improve the quality of the S. rimosus ATCC 10970 genome, producer of oxytetracycline, we validated the assembly of GLPs by applying a new approach to combine pulsed-field gel electrophoresis separation and GLP isolation and sequenced the isolated GLP with Oxford Nanopore technology. By examining the sequenced plasmids of ATCC 10970 and two industrial progenitor strains, R6-500 and M4018, we identified large GLP rearrangements. Analysis of the assembled plasmid sequences shed light on the role of transposases in genome plasticity of this species. The new methodological approach developed for Nanopore sequencing is highly applicable to a variety of sequencing projects. In addition, we present the annotated reference genome sequence of ATCC 10970 with a detailed analysis of the biosynthetic gene clusters.

Community-level and function response of photoautotrophic periphyton exposed to oxytetracycline hydrochloride.

Periphyton is considered important for removal of organic pollutants from water bodies, but knowledge of the impacts of antibiotics on the community structure and ecological function of waterbodies remains limited. In this study, the effects of oxytetracycline hydrochloride (OTC) on the communities of photoautotrophic epilithon and epipelon and its effect on nitrogen and phosphorus concentrations in the water column were studied in a 12-day mesocosm experiment. The dynamics of nitrogen and phosphorus concentrations in the epipelon and epilithon experiment showed similar patterns. The concentrations of total nitrogen, dissolved total nitrogen, ammonium nitrogen, total phosphorus and dissolved total phosphorus in the water column increased rapidly during the initial days of exposure, after which a downward trend occurred. In the epilithon experiment, we found that the photosynthesis (Fv/Fm) and biomass of epilithon were significantly (P < 0.05) stimulated in the low concentration group. Contrarily, growth and photosynthesis (Fv/Fm) were significantly (P < 0.05) reduced in the medium and high concentration group. We further found that the photosynthetic efficiency of photoautotrophic epilithon was negatively correlated with the concentrations of nitrogen and phosphorus in the water column (P < 0.05). Principal coordinate analysis (PCoA) showed that the communities of epilithic algae in the control group and in the low concentration group were significantly (P < 0.05) different from that of the high concentration group during the initial 4 days. After 8 days' exposure, all groups tended to be similar, indicating that epilithon showed rapid adaptability and/or resilience. Similar results were found for the relative abundance of some epilithic algae. Our findings indicate that the biofilm system has strong tolerance and adaptability to OTC as it recovered fast after an initial suppression, thus showing the important role of periphyton in maintaining the dynamic balance of nutrients with other processes in aquatic ecosystems.