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Efflux pumps are a key component in bacteria's ability to gain resistance to antibiotics. In addition to increasing efflux, new research has suggested that the antibiotic, tetracycline, may have larger impacts on bacterial membranes. Using second harmonic scattering, we monitor the transport of two small molecules across the membranes of different Gram-positive bacteria. By comparing our results to a simple kinetic model, we find evidence for changes in influx and efflux across both bacterial species. These changes, however, are probe-dependent, opening new questions about the localization of the drug's effects and the specificity of the efflux pumps involved., (© 2024 Author(s). Published under an exclusive license by AIP Publishing.)

Oxacillin (bactericidal) and tetracycline (bacteriostatic) are clinically relevant antibiotics that are routinely prescribed to treat Staphylococcus aureus infections but not conventionally used in combination. There is an urgent need for treatment regimens that can act upon biofilms during infection, associated with chronic infections on indwelling devices, as well as acute planktonic (systemic) infection. Here we show that in an in vitro model oxacillin and tetracycline act synergistically against S. aureus UAMS-1 biofilms, reducing the concentration of both antibiotics necessary to eradicate an established biofilm. Using an in vivo zebrafish larval infection model with S. aureus NewHG, they display improved bacterial clearance compared to each drug alone and can counteract a loss of host phagocytes, an important innate defence against S. aureus. In these cases, the bacteriostatic nature of tetracycline enhances rather than dampens the bactericidal action of oxacillin, although an exact mechanism remains to be elucidated. We suggest a dual therapy could be of clinical use against biofilm-forming S. aureus and has a potential use in patients with a compromised immune system., (© 2024. The Author(s).)

Litter from cigarette waste is a significant threat to organisms and ecosystems. However, this waste contains cellulose acetate (CA) that can be recycled into raw materials. In this study, recycled CA from cigarettes (CFCA) electrospun through electro-spinning technique and developed hybrid nanocomposite by incorporating CFCA in the fermentation media, followed by self-assembly of bacterial cellulose (BC). CFCA exhibit excessive hydrophobicity due to their high crystallinity and reorientation of hydrophobic groups. We aimed to improve the hydrophilic, thermal and mechanical properties of CFCA. We examined fiber morphology using a scanning electron microscope (SEM), Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction Analysis (XRD), thermogravimetric analysis (TGA), swelling capacity and mechanical properties. BC/CFCA showed higher swelling capacity, improved thermal properties, and good tensile strength compared to CFCA. Additionally, tetracycline hydrochloride (TC) was loaded into developed BC/CFCA matrix and evaluated in-vitro drug release, antibacterial activity and cytotoxicity. In-vitro drug release results showed that developed BC/CFCA can able to control TC release. In addition, prepared BC/CFCA-TC composites demonstrated excellent antibacterial activity against gram-positive and gram-negative bacteria. More importantly, BC/CFCA-TC composites exhibit good cytotoxicity on mouse fibroblast cells (L929). These characteristics of BC/CFCA-TC membranes indicate they may successfully serve as wound dressings and other medical biomaterials., Competing Interests: Declaration of competing interest The authors don't have any conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Salmonella is a major foodborne pathogen that can be transmitted from livestock and poultry to humans through the food chain. Due to the widespread use of antibiotics, antibiotic resistance Salmonella has become an important factor threatening food safety. Combining antibiotic and non-antibiotic agents is a promising approach to address the widespread emergence of antibiotic-resistant pathogens. In this study, we investigated the antibiotic resistance profile and molecular characterization of different serotypes of Salmonella isolated from large-scale egg farms using drug susceptibility testing and whole genome sequencing. The synergistic effect of alpha-linolenic acid (ALA) with antibiotics was evaluated using the checkerboard test and time-kill curve. The molecular mechanism of α-linolenic acid synergism was explored using biochemical assays, pull-down assays, and molecular docking. In vivo efficacy of ALA in combination with florfenicol (FFC) or tetracycline (TET) against multidrug-resistant (MDR) Salmonella enterica subsp. enterica serovar typhimurium was also investigated using a mouse model. We found that ALA reduced the minimum inhibitory concentration (MIC) of tetracycline and florfenicol in all strains tested. When ALA (512 mg/L) was combined with florfenicol (32 mg/L) or tetracycline (16 mg/L), we observed disruption of cell membrane integrity, increased outer membrane permeability, lowered cell membrane potential, and inhibition of proton-drive-dependent efflux pumps. The synergistic treatment also inhibited biofilm production and promoted oxidative damage. These changes together led to an increase in bacterial antibiotic susceptibility. The improved efficacy of ALA combination treatment with antibiotics was validated in the mouse model. Molecular docking results indicate that ALA can bind to membrane proteins via hydrogen bonding. Our findings demonstrated that combined treatment using ALA and antibiotics is effective in preventing infections involving MDR bacteria. Our results are of great significance for the scientific and effective prevention and control of antibiotic resistance Salmonella, as well as ensuring food safety., Competing Interests: Declaration of competing interest This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal. We have read and understood your journal's policies, and we believe that neither the manuscript nor the study violates any of these. There are no conflicts of interest to declare., (Copyright © 2024. Published by Elsevier Ltd.)

The ability to manipulate gene expression is valuable for elucidating gene function. In the fission yeast Schizosaccharomyces pombe, the most widely used regulatable expression system is the nmt1 promoter and its two attenuated variants. However, these promoters have limitations, including a long lag, incompatibility with rich media and unsuitability for non-dividing cells. Here, we present a tetracycline-inducible system free of these shortcomings. Our system features the enotetS promoter, which achieves a similar induced level and a higher induction ratio compared to the nmt1 promoter, without exhibiting a lag. Additionally, our system includes four weakened enotetS variants, offering an expression range similar to that of the nmt1 series promoters but with more intermediate levels. To enhance usability, each promoter is combined with a Tet-repressor-expressing cassette in an integration plasmid. Importantly, our system can be used in non-dividing cells, enabling the development of a synchronous meiosis induction method with high spore viability. Moreover, our system allows for the shutdown of gene expression and the generation of conditional loss-of-function mutants. This system provides a versatile and powerful tool for manipulating gene expression in fission yeast., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Micro- and nanoplastics can interact with various biologically active compounds forming aggregates of which the effects have yet to be understood. To this end, it is vital to characterize these aggregates of key compounds and micro- and nanoplastics. In this study, we examined the adsorption of the antibiotic tetracycline on four different nanoplastics, made of polyethylene (PE), polypropylene (PP), polystyrene (PS), and nylon 6,6 (N66) through chemical computation. Two separate approaches were employed to generate relevant conformations of the tetracycline-plastic complexes. In the first approach, we folded the plastic particle from individual polymer chains in the presence of the drug through multiple separate simulated annealing setups. In the second, more biased, approach, the neat plastic was pre-folded through simulated annealing, and the drug was placed at its surface in multiple orientations. The former approach was clearly superior to the other, obtaining lower energy conformations even with the antibiotic buried inside the plastic particle. Quantum chemical calculations on the structures revealed that the adsorption energies show a trend of decreasing affinity to the drug in the order of N66> PS> PP> PE. In vitro experiments on tetracycline-sensitive cell lines demonstrated that, in qualitative agreement with the calculations, the biological activity of tetracycline drops significantly in the presence of PS particles. Preliminary molecular dynamics simulations on two selected aggregates with each plastic served as first stability test of the aggregates under influence of temperature and in water. We found that all the selected cases persisted in water indicating that the aggregates may be stable also in more realistic environments. In summary, our data show that the interaction of micro- and nanoplastics with drugs can alter drug absorption, facilitate drug transport to new locations, and increase local antibiotic concentrations, potentially attenuating antibiotic effect and at the same time promoting antibiotic resistance., (© 2024. The Author(s).)

Wound care is a flourishing branch of healthcare wherein a great amount of research is devoted to develop competent wound dressings. Safe, cost-effective, and biocompatible dressings aid in wound healing without inflicting external trauma and subsequent scar formation. Toward this, we have attempted to develop robust wound dressing material with self-healing and antibacterial properties. We have cross-linked chitosan with 4-formyl phenylboronic acid (4-FPBA) and in situ generated dehydroascorbic acid (DHA) utilizing the dynamic imine and boronate ester linkages. Displaying a channeled microstructure in the SEM micrographs, the hydrogel exhibits a massive water uptake capacity of ∼900% at acidic pH. The hydrogel could completely self-heal within 3 min, and the results are further supported by rheological analysis. By virtue of positive surface charge, it shows a promising tissue adhesive property. Moreover, it affords clean and compliant removal from the wound surface via dissolution induced by dopamine to potentially reduce secondary scarring from peeling of wound dressings. The dressing could significantly act against skin infections caused by S. aureus bacteria with enhanced antimicrobial efficiency via loading of antibiotic drug, tetracycline hydrochloride. A sustained release of tetracycline and Curcumin was observed, which demonstrated the release ability for hydrophilic and hydrophobic bioactive agents. In-vitro studies revealed 93% cell viability with a hemolytic ratio as low as 2.5%, thereby presenting a good self-healing and biocompatible material for wound healing.

To address the increasing issue of antibiotic wastewater, this study applied a static magnetic field (SMF) to the activated sludge process to increase the efficiency of tetracycline (TC) removal from swine wastewater and to reveal its enhanced mechanisms. The results demonstrated that the SMF-modified activated sludge process could achieve almost complete TC removal at sludge loading rates of 0.3 mg TC/g MLSS/d. Analysis of zeta potential and extracellular polymeric substances composition of the activated sludge revealed that SMF increased electrostatic interactions between TC and activated sludge and made activated sludge has much more binding sites, finally resulting in the increased TC biosorption. Metagenomic analysis showed that SMF promoted the enrichment of ammonia-oxidizing bacteria, TC-degrading bacteria, and aromatic compounds-degrading bacteria; it also enhanced ammonia monooxygenase- and cytochrome P450-mediated TC metabolism while upregulating functional genes associated with oxidase, reductase, and dehydrogenase - all contributing to increased TC biodegradation. Additionally, SMF mitigated the enrichment and spread of antibiotic resistance genes (ARGs) by decreasing the abundance of potential hosts of ARGs and inhibiting the upregulation of genes encoding ABC transporters and putative transposase. Based on these findings, this study demonstrates that magnetic field is an enhancement strategy with great potential to relieve the harmful impacts of the growing antibiotic wastewater problem on human health and the ecosystem., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

The development of nanozymes (NZ) for the simultaneous detection of multiple target chemicals is gaining paramount attention in the field of food and health sciences, and waste management industries. Nanozymes (NZ) effectively compensate for the environmental vulnerability of natural enzymes. Considering the development gap of NZ with diverse applications, we synthesized versatile Schiff's base ligands following a facile route and readily available starting reagents (glutaraldehyde, aminopyridines). DPDI, one of the synthesized ligands, readily reacted with transition metal ions (Cu +2 , Ag +1 , Zn +2 in specific) under ambient conditions, yielding the corresponding nanoparticles/MOF. The structures of ligands and their products were confirmed using various analytical techniques. The enzymatic efficacy of DPDI-Cu (k m 0.25 mM=, V max = 10.75 µM/sec) surpassed Tremetese versicolor laccase efficacy (k m 0. 5 mM=, V max = 2.15 µM/sec). Additionally, DPDI-Cu proved resilient to changing pH, temperature, ionic strength, organic solvent, and storage time compared to laccase and provided reusability. DPDI-Cu proved promising for colorimetric detection of dopamine, epinephrine, catechol, tetracycline, and quercetin. The mechanism of oxidative detection of TC was studied through LC/MS analysis. DPDI-Cu-bentonite composite efficiently adsorbed tetracycline with maximum Langmuir adsorption of 208 mg/g. Moreover, DPDI/Cu and DPDI-Ag nanoparticles possessed antifungal activity exhibiting a minimum inhibitory concentration of 400 µg/mL and 3.12 µg/mL against Aspergillus flavus. Florescent dye tracking and SEM/TEM analysis confirmed that DPDI-Ag caused disruption of the plasma membrane and triggered ROS generation and apoptosis-like death in fungal cells. The DPDI-Ag coating treatment of wheat seeds confirmed the non-phytotoxicity of Ag-NPs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The rise in antibiotic resistance among bacterial pathogens, particularly Staphylococcus aureus , has become a critical global health issue, necessitating the search for novel antimicrobial agents. S. aureus uses various mechanisms to resist antibiotics, including the activation of efflux pumps, biofilm formation, and enzymatic modification of drugs. This study explores the potential of baicalein, a bioflavonoid from Scutellaria baicalensis , in modulating tetracycline resistance in S. aureus by inhibiting efflux pumps. The synergistic action of baicalein and tetracycline was evaluated through various assays. The minimum inhibitory concentration (MIC) of baicalein and tetracycline against S. aureus was 256 and 1.0 μg/mL, respectively. Baicalein at 64 μg/mL reduced the MIC of tetracycline by eightfold, indicating a synergistic effect (fractional inhibitory concentration index: 0.375). Time-kill kinetics demonstrated a 1.0 log CFU/mL reduction in bacterial count after 24 hours with the combination treatment. The ethidium bromide accumulation assay showed that baicalein mediated significant inhibition of efflux pumps, with a dose-dependent increase in fluorescence. In addition, baicalein inhibited DNA synthesis by 73% alone and 92% in combination with tetracycline. It also markedly reduced biofilm formation and the invasiveness of S. aureus into HeLa cells by 52% at 64 μg/mL. These findings suggest that baicalein enhances tetracycline efficacy and could be a promising adjunct therapy to combat multidrug-resistant S. aureus infections.

Antibiotics are frequently employed to control bacterial diseases in honeybees, but their broad-spectrum action can disrupt the delicate balance of the gut microbiome, leading to dysbiosis. This imbalance in the gut microbiota of honeybees adversely affects their physiological health and weakens their resistance to pathogens, including viruses that significantly threaten honeybee health. In this study, we investigated whether tetracycline-induced gut microbiome dysbiosis promotes the replication of Israeli acute paralysis virus (IAPV), a key virus associated with colony losses and whether IAPV infection exacerbates gut microbiome dysbiosis. Our results demonstrated that tetracycline-induced gut microbiome dysbiosis increases the susceptibility of honeybees to IAPV infection. The viral titer in worker bees with antibiotic-induced gut microbiome dysbiosis prior to IAPV inoculation was significantly higher than in those merely inoculated with IAPV. Furthermore, we observed a synergistic effect between tetracycline and IAPV on the disruption of the honeybee gut microbiome balance. The progression of IAPV replication could, in turn, exacerbate antibiotic-induced gut microbiome dysbiosis in honeybees. Our research provides novel insights into the role of the gut microbiota in host-virus interactions, emphasizing the complex interplay between antibiotic use, gut microbiome health, and viral susceptibility in honeybees. We highlight the crucial role of a balanced gut microbiota in honey bees for their immune response against pathogens and emphasize the importance of careful, safe antibiotic use in beekeeping to protect these beneficial microbes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study, a microbial fuel cell was constructed using Raoultella sp. XY-1 to efficiently degrade tetracycline (TC) and assess the effectiveness of the electrochemical system. The degradation rate reached 83.2 ± 1.8 % during the 7-day period, in which the system contained 30 mg/L TC, and the degradation pathway and intermediates were identified. Low concentrations of TC enhanced anodic biofilm power production, while high concentrations of TC decreased the electrochemical activity of the biofilm, extracellular polymeric substances, and enzymatic activities associated with electron transfer. Introducing electrogenic bacteria improved power generation efficiency. A three-strain hybrid system was fabricated using Castellaniella sp. A3, Castellaniella sp. A5 and Raoultella sp. XY-1, leading to the enhanced TC degradation rate of 90.4 % and the increased maximum output voltage from 200 to 265 mV. This study presents a strategy utilizing tetracycline-degrading bacteria as bioanodes for TC removal, while incorporating electrogenic bacteria to enhance electricity generation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Photocatalysts are one of the effective methods to degrade antibiotic contamination, but the efficiency is low and the toxicity is not well recognized. Deep lattice doping to induce defect generation is an effective way to improve the performance of photocatalysts. Here, defect-rich bromine-doped BiOCl-XBr photocatalysts were constructed with the help of small molecules inserted into the interlayer. The photocatalytic degradation performance of BiOCl-XBr was significantly enhanced, and its degradation rate was up to about 12 times that of BiOCl monomer. The main reasons for the stronger photocatalytic performance of BiOCl-XBr include Br doping to enhance visible light absorption, surface defects, and Bi valence changes to improve charge transport. The degradation of tetracycline (TC) produced more toxic intermediates, and the biotoxicity experiments also confirmed that the toxicity showed a trend of increasing and then decreasing, indicating that the more toxic intermediates were also mineralized during the degradation process. However, the mortality and hatching rate of zebrafish in the exposed group after degradation recovered but changed their activity pattern under light and dark conditions. This further warns us to focus on the toxicity changes after antibiotic degradation. Finally, based on the free radical analysis, the mechanism of photocatalytic degradation and detoxification of TC by BiOCl-XBr was proposed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The abuse of tetracycline can lead to its residue in animal derived foods, posing many potential hazards to human health. Therefore, rapid and accurate detection of tetracycline is an important means to ensure food safety. Nitrogen doped and phosphorus doped silicon quantum dots (N-SiQDs, P-SiQDs) with remarkable optical stability were fabricated via a one-pot hydrothermal procedure in this study. Upon the excitation at 346 nm, N-SiQDs and P-SiQDs emitted fluorescence at 431 nm and 505 nm, respectively. Two SiQDs had the potential to serve as a probe for detecting low concentrations of tetracycline (TC), employing a mechanism of the static quenching effect. The calibration curves of N-SiQDs and P-SiQDs were linear within the range of 0-0.8 μM and 0-0.4 μM, the limits of detection were low as 5.35 × 10 -4  μmol/L and 6.90 × 10 -3  μmol/L, respectively. This method could be used successfully to detect TC in honey samples. Moreover, the remarkable antibacterial efficacy of two SiQDs could be attributed to the generation of a large number of intracellular reactive oxygen species. The SEM images showed that the structure of bacterial cell was disrupted and the surface became irregular when treated with both SiQDs. These properties enabled potential usage of SiQDs as excellent antibacterial material for different biomedical applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Alternanthera sessilis (AS) leaf extract was used to synthesize zinc oxide nanoparticles (ZnO NPs). Bioanalytical characterization techniques such as X-ray diffraction (XRD) and field emission scanning electron microscope (FESEM) confirmed the formation of crystalline ZnO NPs with average sizes of 40 nm. The AS-ZnO NPs antimicrobial activity was analyzed under dark (D) and white light (WL) conditions. The improved antimicrobial activity was observed against Escherichia coli, Staphylococcus aureus and Bacillus subtilis at the minimal inhibitory concentration (MIC) of 125 and 62.5 µg/mL under WL than the D at 125 and 250 µg/mL for E. coli, B. subtilis, and Pseudomonas aeruginosa, respectively. In contrast, the growth of P. aeruginosa and S. aureus was not completely inhibited until 1 mg/mL AS-ZnO NPs under WL and D. Similarly, AS-ZnO NPs displayed a weaker inhibitory effect against carbapenem-sensitive P. aeruginosa (CSPA) and carbapenem-resistant P. aeruginosa (CRPA) strains of PAC023, PAC041 and PAC032, PAC045 under D. Interestingly, the distinct inhibitory effect was recorded against CSPA PAC041 and CRPA PAC032 in which the bacteria growth was inhibited 99.9% at 250, 500 µg/mL under WL. The cytotoxicity results suggested AS-ZnO NPs demonstrated higher toxicity to MCF-7 breast cancer cells than the RAW264.7 macrophage cells. Further, AS-ZnO NPs exhibited higher catalytic potential against tetracycline hydrochloride (TC-H) degradation at 65.6% and 60.8% under WL than the dark at 59.35% and 48.6% within 120 min. Therefore, AS-ZnO NPs can be used to design a photo-improved antimicrobial formulation and environmental catalyst for removing TC-H from wastewater., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Neisseria gonorrhoeae (Ng) is a public health priority because of the rapid evolution of antimicrobial resistance, the emergence of antibiotic resistance, and the absence of a vaccine against Ng. The aim of this study was to investigate trends in the minimum inhibitory concentration and resistance (R) or reduced susceptibility (DS) of Ng cases to ceftriaxone (CRO), azithromycin (AZM), tetracycline (TET), benzylpenicillin (PenG), and ciprofloxacin (CIP) during a 10-year period., Methods: We conducted a retrospective analysis on an open cohort of Ng cases diagnosed on rectal, urethral, and pharyngeal samples at San Raffaele Scientific Institute, between September 2012 and February 2023. Minimum inhibitory concentrations of antibiotics were determined by gradient-test strips. Bivariate linear regression models were applied on logarithmic minimum inhibitory concentrations values; Cochran-Armitage test was used to determine a linear trend in the proportions of resistant strains., Results: A total of 436 Ng isolates from 352 individuals were analyzed. Minimum inhibitory concentrations of CRO and PenG reduced over time ( P < 0.001, P = 0.030), AZM increased ( P = 0.001), and CIP and TET did not change ( P = 0.473, P = 0.272). The percentages of resistant strains were as follows: PenG, 89.9%; TET, 90.8%; CIP, 48.2%; AZM, and 4.4%. CRO-DS strains were 8.7%, and only 1 case of CRO-R was identified. The proportion of resistant strains increased over time for AZM ( P = 0.007), TET ( P = 0.001), and CIP ( P < 0.001), whereas it decreased for PenG ( P < 0.001) and CRO-DS/R strains ( P < 0.001)., Conclusions: Ng strains showed high susceptibility to CRO, although we identified cases of DS/R and observed high levels of susceptibility to AZM. Overall, the recommended primary regimen for Ng treatment was confirmed to be effective., Competing Interests: Conflict of Interest and Source of Funding: None declared., (Copyright © 2024 American Sexually Transmitted Diseases Association. All rights reserved.)

The new water-soluble di-anionic bi-sodium salt of tetracycline (TC), an antibiotic in clinical use, with the formula {[TC] 2- [Na + (MeOH)(H 2 O)] [Na + ]·(H 2 O)} n (TCNa) was synthesized. The compound was characterized by m.p., attenuated total reflectance-Fourier transform infra-red (ATR-FTIR) spectroscopy, and ultraviolet (UV) and proton nuclear magnetic resonance ( 1 H NMR) spectroscopy in the solid state and in solution. The molecular weight (MW) was determined by cryoscopy. The crystal structure of TCNa was also determined by X-ray crystallography. The antibacterial activity of TCNa was evaluated against the bacterial species Pseudomonas aeruginosa ( P. aeruginosa ), Escherichia coli ( E. coli ), Staphylococcus epidermidis ( S. epidermidis ) and Staphylococcus aureus ( S. aureus ) by means of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and inhibition zones (IZs). Moreover, the ability of the compound to eradicate biofilm formation was also evaluated. The results are compared with those obtained for the commercially available drug TCH2. The in vitro and in vivo toxicities of TCNa were tested against human corneal epithelial cells (HCECs) and Artemia salina .

Tetracycline and copper ion are common pollutants in wastewater, and the effects of mixed pollutants on microorganisms in wastewater biological treatment have been less studied. In order to reveal the effects of mixed pollutants of tetracycline and copper ion on the microorganisms during the biological phosphorus removal, three ratios of tetracycline and copper ions were designed by the direct equipartition ray method. The relative abundance and diversity of microbial community were investigated, and the microbial interactions were revealed through microbiological methods. The results demonstrated that, for three different ratios, the inhibitory effect of specific phosphorus uptake rate became more significant with the increase of the tetracycline-copper ions concentration and the reaction time. The microbial community decreased with the increase of the proportion of tetracycline in different ratios. The relative abundance of Acinetobacter decreased with the increase of the proportion of tetracycline, while the relative abundance of Ca.Competibacter was higher under the conditions of low mixtures concentrations. Positive interactions and symbiotic relationships among microorganisms were predominant for three different ratios. However, as the proportion of tetracycline increased, the community structure of microorganisms shifted from phosphate-accumulating organisms to glycogen accumulating organisms and denitrifying bacteria. This study can provide a reference for the effect of mixed pollutants on microorganisms and the mechanism of wastewater treatment., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Minimum inhibitory concentrations (MIC) assays are often questioned for their representativeness. Especially when foodborne pathogens are tested, it is of crucial importance to also consider parameters of the human digestive system. Hence, the current study aimed to assess the inhibitory capacity of two antibiotics, ciprofloxacin and tetracycline, against Salmonella enterica and Listeria monocytogenes, under representative environmental conditions. More specifically, aspects of the harsh environment of the human gastrointestinal tract (GIT) were gradually added to the experimental conditions starting from simple aerobic lab conditions into an in vitro simulation of the GIT. In this way, the effects of parameters including the anoxic environment, physicochemical conditions of the GIT (low gastric pH, digestive enzymes, bile acids) and the gut microbiota were evaluated. The latter was simulated by including a representative consortium of selected gut bacteria species. In this study, the MIC of the two antibiotics against the relevant foodborne pathogens were established, under the previously mentioned environmental conditions. The results of S. enterica highlighted the importance of the anaerobic environment when conducting such studies, since the pathogen thrived under such conditions. Inclusion of physicochemical barriers led to exactly opposite results for S. enterica and L. monocytogenes since the former became more susceptible to ciprofloxacin while the latter showed lower susceptibility towards tetracycline. Finally, the inclusion of gut bacteria had a bactericidal effect against L. monocytogenes even in the absence of antibiotics, while gut bacteria protected S. enterica from the effect of ciprofloxacin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

The metabolic environment is responsible for antibiotic resistance, which highlights the way in which the antibiotic resistance mechanism works. Here, GC-MS-based metabolomics with iTRAQ-based proteomics was used to characterize a metabolic state in tetracycline-resistant Escherichia coli K12 ( E. coli -R TET ) compared with tetracycline-sensitive E. coli K12. The repressed pyruvate cycle against the elevation of the proton motive force (PMF) and ATP constructed the most characteristic feature as a consequence of tetracycline resistance. To understand the role of the elevated PMF in tetracycline resistance, PMF inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and the pH gradient were used to investigate how the elevation influences bacterial viability and intracellular antibiotic concentration. A strong synergy was detected between CCCP and tetracycline to the viability, which was consistent with increasing intracellular drug and decreasing external pH. Furthermore, E. coli -R TET and E. coli -R GEN with high and low PMF concentrations were susceptible to gentamicin and tetracycline, respectively. The elevated PMF in E. coli -R TET was attributed to the activation of other metabolic pathways, except for the pyruvate cycle, including a malate-oxaloacetate-phosphoenolpyruvate-pyruvate-malate cycle. These results not only revealed a PMF-dependent mechanism for tetracycline resistance but also provided a solution to tetracycline-resistant pathogens by aminoglycosides and aminoglycoside-resistant bacteria by tetracyclines.

Ribosomes translate the genetic code into proteins. Recent technical advances have facilitated in situ structural analyses of ribosome functional states inside eukaryotic cells and the minimal bacterium Mycoplasma. However, such analyses of Gram-negative bacteria are lacking, despite their ribosomes being major antimicrobial drug targets. Here we compare two E. coli strains, a lab E. coli K-12 and human gut isolate E. coli ED1a, for which tetracycline exhibits bacteriostatic and bactericidal action, respectively. Using our approach for close-to-native E. coli sample preparation, we assess the two strains by cryo-ET and visualize their ribosomes at high resolution in situ. Upon tetracycline treatment, these exhibit virtually identical drug binding sites, yet the conformation distribution of ribosomal complexes differs. While K-12 retains ribosomes in a translation-competent state, tRNAs are lost in the vast majority of ED1a ribosomes. These structural findings together with the proteome-wide abundance and thermal stability assessments indicate that antibiotic responses are complex in cells and can differ between different strains of a single species, thus arguing that all relevant bacterial strains should be analyzed in situ when addressing antibiotic mode of action., (© 2024. The Author(s).)

Drug efflux systems have recently been recognized as a significant mechanism responsible for multidrug resistance in bacteria. In this study, we described the identification and characterization of a new chromosomally encoded efflux pump (SA00565) in Staphylococcus aureus . SA00565, which belongs to the drug/metabolite transporter (DMT) superfamily, was predicted to be a 10-transmembrane segment transporter. To evaluate the role of sa00565 in resistance, we generated sa00565 gene deletion mutant (Δ sa00565 ) and assessed its susceptibility to 35 different antibiotic treatments. Our results demonstrated that the Δ sa00565 mutant exhibited reduced resistance to tetracycline and doxycycline, with 64-fold and 12-fold decreased MICs, respectively. The mechanism of SA00565-mediated tetracycline resistance was demonstrated that SA00565 possesses the capability to efficiently extrud intracellular tetracycline into the environment. The efflux activity of SA00565 was further validated using EtBr accumulation and efflux assays. In summary, our study uncovered a previously unknown function of a DMT family transporter, which serves as a tetracycline efflux pump, thereby contributing to tetracycline resistance in S. aureus .IMPORTANCEIn this study, we addressed the significance of drug efflux systems in multidrug resistance of Staphylococcus aureus , focusing on the unexplored efflux pump SA00565 in the drug/metabolite transporter (DMT) superfamily. Through phylogenetic analysis, gene knockout, and overexpression experiments, we identified the role of SA00565 in antibiotic resistance. The Δ sa00565 mutant showed increased susceptibility to tetracycline and doxycycline in disk diffusion assays, with significantly lower MICs compared to the WT. Remarkably, intracellular tetracycline concentration in the mutant was two- to threefold higher, indicating SA00565 actively eliminates intracellular tetracycline. Our findings emphasize the pivotal contribution of SA00565 to tetracycline antibiotic resistance in S. aureus , shedding light on its functional attributes within the DMT superfamily and providing valuable insights for combating multidrug resistance., Competing Interests: The authors declare no conflict of interest.

In this research, poly-chloropropylmethyl-silsesquioxanen was prepared and decorated with ZIF-8 in order to investigate its loading capacity for acyclovir and tetracycline. Before and after drug loadings, the composites were characterized by FT-IR, SEM-EDS, XRD, and XPS analyses. Then, the in-vitro release of these drugs was investigated by UV-Vis spectroscopy in different buffers (pH = 5, 7.4, and 9.1). The results showed that the release of ACV reached a maximum amount of 41.3 mg at pH = 7.4 during 12 h. In comparison, the release of TC reached a maximum amount of 22.5 mg at pH = 5 during 6 h. The blood compatibility, in-vitro cytotoxicity on the L929 fibroblast cells line, and antibacterial assay against Staphylococcus aureus and Pseudomonas aeruginosa were also investigated for this composite as a drug carrier., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Triclosan (TCS) and tetracycline (TC) are commonly detected antibacterial agents in sewage and environment matrices. Nonetheless, the impact of sequential exposure to TCS and TC on the methanogenic digestion microbiome remains unknown. In this study, TCS was shown to reduce COD removal efficiency to 69.8%, but alleviated the inhibitive effect of consequent TC-amendment on the digestion microbiome. Interestingly, TCS pre-exposure resulted in abundance increase of acetotrophic Methanosaeta to 2.68%, being 2.91 folds higher than that without TCS amendment. Microbial network analyses showed that TCS pre-exposure caused microorganisms to establish a co-ecological relationship against TC disturbance. Further analyses of total antibiotic resistance genes (ARGs) showed the TCS-derived compromise of TC-induced ARGs enrichment in digestion microbiomes, e.g., 238.2% and 152.1% ARGs increase upon TC addition in digestion microbiomes without and with TCS pre-exposure, respectively. This study provides new insights into the impact of antibacterial agents on the methanogenic digestion microbiome., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Diarrhoea remains an important public health concern, particularly in developing countries, and has become difficult to treat because of antibacterial resistance. The development of synergistic antimicrobial agents appears to be a promising alternative treatment against diarrhoeic infections. In this study, the combined effect of tetracycline together with either nitroxoline, sanguinarine, or zinc pyrithione (representing various classes of plant-based compounds) was evaluated in vitro against selected diarrhoeic bacteria ( Enterococcus faecalis , Escherichia coli , Listeria monocytogenes , Shigella flexneri , Vibrio parahaemolyticus , and Yersinia enterocolitica ). The chequerboard method in 96-well microtiter plates was used to determine the sum of the fractional inhibitory concentration indices (FICIs). Three independent experiments were performed per combination, each in triplicate. It was observed that the combination of tetracycline with either nitroxoline, sanguinarine, or zinc pyrithione produced synergistic effects against most of the pathogenic bacteria tested, with FICI values ranging from 0.086 to 0.5. Tetracycline-nitroxoline combinations produced the greatest synergistic action against S. flexneri at a FICI value of 0.086. The combinations of the agents tested in this study can thus be used for the development of new anti-diarrhoeic medications. However, studies focusing on their in vivo anti-diarrhoeic activity and safety are required before any consideration for utilization in human medicine.

Tetracycline exerts an inhibitory effect on anaerobic digestion, inducing stressed microbial activities and even system failure. Continuous-flow reactors (CFRs) and sequencing batch reactors (SBRs) were employed along with the dosage of powdered activated carbon (PAC) to enhance tetracycline removal during anaerobic digestion of complex organic compounds. PAC increased the maximum methane production rate by 15.6% (CFRs) and 13.8% (SBRs), and tetracycline biodegradation by 24.4% (CFRs) and 19.2% (SBRs). CFRs showed higher tetracycline removal and methane production rates than SBRs. Geobacter was enriched in CFRs, where Methanothrix was enriched with the addition of PAC. Desulfomicrobium harbored abundant propionate degradation-related genes, significantly correlating with tetracycline removal. The genes encoding carbon dioxide reduction in Methanothrix along with the detection of Geobacter might indicate direct interspecies electron transfer for methanogenesis in CFRs and PAC-added reactors. The study offers new insights into anaerobic digestion under tetracycline-stressed conditions and strategies for optimizing tetracycline removal., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Guangxue Wu reports financial support was provided by Sustainable Energy Authority of Ireland. Yuyin Wang reports financial support was provided by China Scholarship Council. Guangxue Wu reports financial support was provided by Galway University Foundation CLG. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Fungal laccase has strong ability in detoxification of many environmental contaminants. A putative laccase gene, LeLac12, from Lentinula edodes was screened by secretome approach. LeLac12 was heterogeneously expressed and purified to characterize its enzymatic properties to evaluate its potential use in bioremediation. This study showed that the extracellular fungal laccase from L. edodes could effectively degrade tetracycline (TET) and the synthetic dye Acid Green 25 (AG). The growth inhibition of Escherichia coli and Bacillus subtilis by TET revealed that the antimicrobial activity was significantly reduced after treatment with the laccase-HBT system. 16 transformation products of TET were identified by UPLC-MS-TOF during the laccase-HBT oxidation process. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that LeLac12 could completely mineralize ring-cleavage products. LeLac12 completely catalyzed 50 mg/L TET within 4 h by adding AG (200 mg/L), while the degradation of AG was above 96% even in the co-contamination system. Proteomic analysis revealed that central carbon metabolism, energy metabolism, and DNA replication/repair were affected by TET treatment and the latter system could contribute to the formation of multidrug-resistant strains. The results demonstrate that LeLac12 is an efficient and environmentally method for the removal of antibiotics and dyes in the complex polluted wastewater., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hao Yu, Chunhui Hu, Jingtian Hu, Shuxue Zhao has patent pending to 2023104452844. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Tigecycline is an antibiotic of last resort for infections with carbapenem-resistant Acinetobacter baumannii . Plasmids harboring variants of the tetracycline destructase gene tetX promote rising tigecycline resistance rates. We report the earliest observation of tet(X3 ) in a clinical strain predating tigecycline's commercialization, suggesting selective pressures other than tigecycline contributed to its emergence., Importance: We present the earliest observation of a tet ( X3 )-positive bacterial strain, predating by many years the earliest reports of this gene so far. This finding is significant as tigecycline is an antibiotic of last resort for carbapenem-resistant Acinetobacter baumannii (CRAB), which the World Health Organization ranks as one of its top three critical priority pathogens, and tet ( X3 ) variants have become the most prevalent genes responsible for enabling CRAB to become tigecycline resistant. Moreover, the tet ( X3 )-positive strain we report is the first and only to be found that predates the commercialization of tigecycline, an antibiotic that was thought to have contributed to the emergence of this resistance gene. Understanding the factors contributing to the origin and spread of novel antibiotic resistance genes is crucial to addressing the major global public health issue, which is antimicrobial resistance., Competing Interests: The authors declare no conflict of interest.

Novel hydrogel beads based on nanocomposite with outstanding antibacterial and swelling capabilities have been successfully produced as an efficient drug carrier for potential drug delivery systems in wound healing applications. The beads were characterized by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and EDX-Mapping analysis. Then, using tetracycline hydrochloride (TCH) as a model drug system, they were studied in vitro for their potential efficiency as pH and temperature dependent sustained drug delivery carriers. Moreover, they were assessed in terms of porosity, swelling degree, encapsulation efficiency, and in vitro release kinetics. Beads released drugs at their highest levels under alkaline circumstances (pH = 8) and at a temperature of 39 °C, with a cumulative TCH release of 96.2 % at 36 h and in accordance with the Weibull kinetics model (R 2  = 0.98). Additionally, the disc diffusion experiment demonstrated the strong antibacterial activity of the synthesized beads and offered a feasible and cost-effective wound dressing material for treating infected wounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The widespread use of copper and tetracycline as growth promoters in the breeding industry poses a potential threat to environmental health. Nevertheless, to the best of our knowledge, the potential adverse effects of copper and tetracycline on the gut microbiota remain unknown. Herein, mice were fed different concentrations of copper and/or tetracycline for 6 weeks to simulate real life-like exposure in the breeding industry. Following the exposure, antibiotic resistance genes (ARGs), potential pathogens, and other pathogenic factors were analyzed in mouse feces. The co-exposure of copper with tetracycline significantly increased the abundance of ARGs and enriched more potential pathogens in the gut of the co-treated mice. Copper and/or tetracycline exposure increased the abundance of bacteria carrying either ARGs, metal resistance genes, or virulence factors, contributing to the widespread dissemination of potentially harmful genes posing a severe risk to public health. Our study provides insights into the effects of copper and tetracycline exposure on the gut resistome and potential pathogens, and our findings can help reduce the risks associated with antibiotic resistance under the One Health framework., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Tetracycline destructases (TDases) are flavin monooxygenases which can confer resistance to all generations of tetracycline antibiotics. The recent increase in the number and diversity of reported TDase sequences enables a deep investigation of the TDase sequence-structure-function landscape. Here, we evaluate the sequence determinants of TDase function through two complementary approaches: (1) constructing profile hidden Markov models to predict new TDases, and (2) using multiple sequence alignments to identify conserved positions important to protein function. Using the HMM-based approach we screened 50 high-scoring candidate sequences in Escherichia coli, leading to the discovery of 13 new TDases. The X-ray crystal structures of two new enzymes from Legionella species were determined, and the ability of anhydrotetracycline to inhibit their tetracycline-inactivating activity was confirmed. Using the MSA-based approach we identified 31 amino acid positions 100% conserved across all known TDase sequences. The roles of these positions were analyzed by alanine-scanning mutagenesis in two TDases, to study the impact on cell and in vitro activity, structure, and stability. These results expand the diversity of TDase sequences and provide valuable insights into the roles of important residues in TDases, and flavin monooxygenases more broadly., (© 2024. The Author(s).)

Tetracyclines serve as broad-spectrum antibiotics to treat bacterial infections. The discovery of new tetracycline resistance genes has led to new questions about the underlying mechanisms of resistance, gene transfer, and their relevance to human health. We tracked changes in the abundance of a 55-kbp conjugative transposon (CTn214) carrying tetQ, a tetracycline resistance gene, within a Bacteroides fragilis metagenome-assembled genome derived from shotgun sequencing of microbial DNA extracted from the ileal pouch of a patient with ulcerative colitis. The mapping of metagenomic reads to CTn214 revealed the multi-copy nature of a 17,044-nt region containing tetQ in samples collected during inflammation and uninflamed visits. B. fragilis cultivars isolated from the same patient during periods of inflammation harbored CTn214 integrated into the chromosome or both a circular, multi-copy, extrachromosomal region of the CTn214 containing tetQ and the corresponding integrated form. The tetracycline-dependent mechanism for the transmission of CTn214 is nearly identical to a common conjugative transposon found in the genome of B. fragilis (CTnDOT), but the autonomously amplified nature of a circular 17,044-nt region of CTn214 that codes for tetQ and the integration of the same sequence in the linear chromosome within the same cell is a novel observation. Genome and transcriptome sequencing of B. fragilis cultivars grown under different concentrations of tetracycline and ciprofloxacin indicates that tetQ in strains containing the circular form remains actively expressed regardless of treatment, while the expression of tetQ in strains containing the linear form increases only in the presence of tetracycline.IMPORTANCEThe exchange of antibiotic production and resistance genes between microorganisms can lead to the emergence of new pathogens. In this study, short-read mapping of metagenomic samples taken over time from the illeal pouch of a patient with ulcerative colitis to a Bacteroides fragilis metagenome-assembled genome revealed two distinct genomic arrangements of a novel conjugative transposon, CTn214, that encodes tetracycline resistance. The autonomous amplification of a plasmid-like circular form from CTn214 that includes tetQ potentially provides consistent ribosome protection against tetracycline. This mode of antibiotic resistance offers a novel mechanism for understanding the emergence of pathobionts like B. fragilis and their persistence for extended periods of time in patients with inflammatory bowel disease., Competing Interests: The authors declare no conflict of interest.

Antibiotic abuse is the main reason for the drug resistance of pathogenic bacteria, posing a potential health risk. Antibiotic surveillance is critical for preventing antibiotic contamination. This study aimed to develop a sensitive and broad-spectrum whole-cell biosensor for tetracycline antibiotics (TCs) detection. Wild-type TCs-responsive biosensor was constructed by introducing a tetracycline operon into a sfGFP reporter plasmid. Using error-prone PCR, mutation libraries containing approximately 10 7 variants of the tetracycline repressor (TetR) gene were generated. The tigecycline-senstive mutants were isolated using high-throughput flow cytometric sorting. After 2 rounds of directed evolution, a mutant epS2-22 of TerR was isolated and assembled as a TCs biosensor. The epS2-22 biosensor was more sensitive and broad-spectrum than the wild-type biosensors. The detection limits of the epS2-22 biosensor for seven TCs were 4- to 62-fold lower than the wild-type biosensor (no response to tigecycline). Meanwhile, the epS2-22 biosensor had a shorter detection time and a stronger signal output than the wild type. In addition, the evolved epS2-22 biosensor showed excellent performance in detecting low traces of TCs in environmental water. These results suggest that directed evolution is a powerful tool for developing high-performance whole-cell biosensors, and the evolved epS2-22 biosensors have the potential for wider applications in real-world TCs detection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

We report data consistent with tetracycline-mediated fluorescence having the potential to be an effective marker of senescence in immortalised cells. HeLa cells that had previously undergone more than 20 passages were transiently transfected with a plasmid encoding a novel tetracycline-inducible transgene featuring an open reading frame for green fluorescent protein. While characterising the performance of this plasmid and transfection procedure, HeLa cell fluorescence was observed to result from incubating cells with media containing 2 μg/ml tetracycline alone, without plasmid or transfection reagent. To investigate this phenomenon further, HeLa and HEK293T cells were purchased from a tissue culture collection and after cultivation over 4-23 passages, incubated with media containing 2 μg/ml tetracycline. For both cell lines, tetracycline-mediated fluorescence increase correlated with passage number increase. This effect in HeLa and HEK293T cells was also borne out by expression of β-galactosidase activity, an imperfect but widely used marker of cellular senescence. These data suggest tetracycline may have utility as a marker of cellular senescence in immortal cells and can inform future investigation and validation of this previously unreported application for this reagent., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Non-tuberculous mycobacterial infections are gradually increasing worldwide, with slow-growing mycobacteria such as Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium kansasii accounting for the majority of cases. The use of tetracyclines has received renewed attention in recent years, and this study was designed to investigate the antibacterial activity of omadacycline, eravacycline, tigecycline, sarecycline, minocycline and doxycycline against M. avium, M. intracellulare and M. kansasii. Susceptibility testing of six tetracyclines was conducted against M. avium, M. intracellulare and M. kansasii isolates, and all the clinical isolates were collected from January 2012 to December 2018. All six agents exhibited poor antibacterial activity against slowly growing mycobacteria (SGM) isolates of three subspecies with MIC 50 and MIC 90  ≥8 μg/mL. M. intracellulare and M. kansasii had lower resistance rates to omadacycline than the other five drugs. The severe resistance of SGM to tetracycline suggests that developing tetracycline-class antibiotics needs to overcome existing resistance mechanisms., (© 2023 John Wiley & Sons Australia, Ltd.)

In this study, activated carbon-loaded nano-zero-valent iron (nZVI-C) composites were added to anaerobic ammonium oxidation bacteria (AnAOB) to overcome the inhibition of tetracycline hydrochloride (TCH). Results showed that 500 mg L -1 nZVI-C effectively mitigated the long-term inhibition of 1.5 mg L -1 TCH on AnAOB and significantly improved the total nitrogen removal efficiency (TNRE) (from 65.27% to 86.99%). Spectroscopic analysis revealed that nZVI-C increased the content of N-H and CO groups in EPS, which contributed to the adsorption of TCH. The accumulation of humic acid-like substances in EPS was also conducive to strengthening the extracellular defense level. In addition, TCH-degrading bacteria (Clostridium and Mycobacterium) were enriched in situ, and the abundance of Ca. Brocadia was significantly increased (from 10.69% to 18.59%). Furthermore, nZVI-C increased the abundance of genes encoding tetracycline inactivation (tetX), promoted mineralization of TCH by 90%, weakening the inhibition of TCH on microbial nitrogen metabolism. nZVI-C accelerated the electron consumption of anammox bacteria by upregulating the abundance of electron generation genes (nxrA, hdh) and providing electrons directly., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Inactivation of tetracycline antibiotics by tetracycline destructases (TDases) remains a clinical and agricultural threat. TDases can be classified as type 1 Tet(X)-like TDases and type 2 soil-derived TDases. Type 1 TDases are widely identified in clinical pathogens. A combination therapy of tetracycline and a TDase inhibitor is much needed to rescue the clinical efficacy of tetracyclines. Anhydrotetracycline is a pan-TDase inhibitor that inhibits both type 1 and type 2 TDases. Here, we present structural, biochemical, and phenotypic evidence that anhydrotetracycline binds in a substrate-like orientation and competitively inhibits the type 1 TDase Tet(X6) to rescue tetracycline antibiotic activity as a sacrificial substrate. Anhydrotetracycline interacting residues of Tet(X6) are conserved within type 1 TDases, indicating a conserved binding mode and mechanism of inhibition. This mode of binding and inhibition is distinct from anhydrotetracycline's inhibition of type 2 TDases. This study forms the framework for development of next-generation therapies to counteract enzymatic tetracycline resistance., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Tetracyclines (TCs) are an important class of antibiotics threatened by an emerging new resistance mechanism─enzymatic inactivation. These TC-inactivating enzymes, also known as tetracycline destructases (TDases), inactivate all known TC antibiotics, including drugs of last resort. Combination therapies consisting of a TDase inhibitor and a TC antibiotic represent an attractive strategy for overcoming this type of antibiotic resistance. Here, we report the structure-based design, synthesis, and evaluation of bifunctional TDase inhibitors derived from anhydrotetracycline (aTC). By appending a nicotinamide isostere to the C9 position of the aTC D-ring, we generated bisubstrate TDase inhibitors. The bisubstrate inhibitors have extended interactions with TDases by spanning both the TC and presumed NADPH binding pockets. This simultaneously blocks TC binding and the reduction of FAD by NADPH while "locking" TDases in an unproductive FAD "out" conformation.

This study analyzed the effect of high-pressure processing (HPP) on the frequency of conjugal gene transfer of antibiotic resistance genes among strains obtained from starter cultures. Gene transfer ability was analyzed in vitro and in situ in the food matrix. It was found that the transfer of aminoglycoside resistance genes did not occur after high-pressure treatment, either in vitro or in situ. After exposure to HPP, the transfer frequencies of tetracycline, ampicillin and chloramphenicol resistance genes increased significantly compared to the control sample, both in vitro and in situ. The frequency of resistance genes transfer in the food matrix in the pressurized samples did not differ significantly from the in vitro transfer rate. Minimum Inhibitory Concentrations (MICs) for these antibiotics determined for transconjugants were lower or equal to MICs determined for the donors. No significant differences were observed between the MIC values determined for the transconjugants obtained in vitro and in situ. The results suggest that HPP may contribute to the spread of antibiotic resistance. This points to the need to verify starter cultures strains for their antibiotic resistance and pressurization parameters to avoid spreading antibiotic resistance genes., Competing Interests: Declaration of competing interest Authors declared no conflict of interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Due to its high instability and rapid degradation under adverse conditions, tetracycline hydrochloride (TC) can cause difficulties in the development of an effective but stable formulation for the topical treatment of acne. The aim of the following work was to propose a hydrogel formulation that would ensure the stability of the antibiotic contained in it. Additionally, an important property of the prepared formulations was the activity of the alcoholamines contained in them against the components of the model sebum. This feature may help effectively cleanse the hair follicles in the accumulated sebum layer. A series of formulations with varying proportions of anionic polymer and alcoholamine and containing different polymers have been developed. The stability of tetracycline hydrochloride contained in the hydrogels was evaluated for 28 days by HPLC analysis. Formulations containing a large excess of TRIS alcoholamine led to the rapid degradation of TC from an initial concentration of about 10 µg/mL to about 1 µg/mL after 28 days. At the same time, these formulations showed the highest activity against artificial sebum components. Thanks to appropriately selected proportions of the components, it was possible to develop a formulation that assured the stability of tetracycline for ca. one month, while maintaining formulation activity against the components of model sebum.

The widespread use of antibiotics has led to a large number of antibiotics entering the environment, to which microorganisms have become resistant. In recent years, with the intensification of human activities in the plateau region, the occurrence and migration of antibiotic resistance genes (ARGs) in plateau wetlands have attracted considerable attention. Here, we selected the Caohai National Wetland Park, located in the Yunnan-Guizhou Plateau, as our study area. The contents of tetracyclines, sulfonamides, quinolones, and macrolides in sediments from the upstream (the pristine habitat near the spring eye) and downstream (the sewage discharge outlet of residents) areas of the river in the park were analyzed. Among them, the detection content of tetracycline antibiotics was 103.65-2185 μg·kg -1 , which was the highest antibiotic detection content. To further investigate the occurrence characteristics and influencing factors of tetracycline resistance genes, the influence of environmental factors, bacterial community structure, and pathogenic bacteria on tetracycline ARGs under the influence of human activities were revealed via correlation analysis and network analysis. The results showed that a total of 15 tetracycline resistance genes were detected in the upstream and downstream sediments. Among them, seven resistance genes including tetPA , tetD , and tetPB were detected in the upstream, and 13 resistance genes such as tetPA , tetE , tetM , and tetX were detected in the downstream. The abundance of eight new resistance genes in the downstream accounted for 43.44% of the downstream genes. The tetracycline-like antibiotics and soil physicochemical indicators (i.e., available phosphorus, total organic carbon, nitrate nitrogen, and total phosphorus) were the main environmental factors affecting the distribution of tetracycline ARGs. Additionally, the bacteria detected in the upstream and downstream sediments belonged to 64 bacterial phyla, among which Proteobacteria, Firmicutes, and Bacteroidota were the main phyla affecting the abundance of tetracycline ARGs; meanwhile, 27 pathogenic bacteria were detected in the upstream and downstream sediments. Network analysis showed that the correlation between the eight new resistance genes and pathogens in the downstream area accounted for 70% of the network connectivity, and Listeria monocytogenes , Enterococcus faecalis , and Bacteroides vulgatus were identified as potential hosts for the transmission of tetracycline ARGs. Compared to the pristine habitat, the discharge of domestic sewage introduced large amounts of antibiotics and also changed the microenvironment and microbial community structure of the river wetland. Additionally, it increased the species of ARGs in sediments, which promoted the spread and transmission of ARGs among microorganisms and even pathogens.

Inducible promoters are indispensable elements when considering the possibility to modulate gene expression on demand. Desirable traits of conditional expression systems include their capacity for tight downregulation, high overexpression, and in some instances for fine-tuning, to achieve a desired product's stoichiometry. Although the number of inducible systems is slowly increasing, suitable promoters comprising these features are rare. To date, the concomitant use of multiple regulatable promoter platforms for controlled multigene expression has been poorly explored. This work provides pioneer work in the human pathogenic fungus Aspergillus fumigatus, wherein we investigated different inducible systems, elucidated three candidate promoters, and proved for the first time that up to three systems can be used simultaneously without interfering with each other. Proof of concept was obtained by conditionally expressing three antifungal drug targets within the ergosterol biosynthetic pathway under the control of the xylose-inducible PxylP system, the tetracycline-dependent Tet-On system, and the thiamine-repressible PthiA system. IMPORTANCE In recent years, inducible promoters have gained increasing interest for industrial or laboratory use and have become key instruments for protein expression, synthetic biology, and metabolic engineering. Constitutive, high-expressing promoters can be used to achieve high expression yields; however, the continuous overexpression of specific proteins can lead to an unpredictable metabolic burden. To prevent undesirable effects on the expression host's metabolism, the utilization of tunable systems that allow expression of a gene product on demand is indispensable. Here, we elucidated several excellent tunable promoter systems and verified that each can be independently induced in a single strain to ultimately develop a unique conditional multigene expression system. This highly efficient, modular toolbox has the potential to significantly advance applications in fundamental as well as applied research in which regulatable expression of several genes is a key requirement.

Tetracycline (TC) antibiotic removal from water bodies is important to provide clean water and sanitation. Mesoporous graphitic carbon nitride (GCN) photocatalyst derived from three different types of precursors manages to remove TC effectively under visible light irradiation. Among urea, thiourea, and melamine precursors, melamine-prepared GCN (MGCN) via thermal polymerization has the highest efficiency to photodegrade tetracycline (TC) antibiotics up to 99.5% (0.0122 min -1 ) within 240 min. The COD for TC removal by using MGCN was up to 77.5% after 240 min of degradation. This is due to the slow charge recombination and rapid charge carrier migration. The MGCN encounters different properties such as high crystallinity, dense structure allowing fast charges migration, and nitrogen vacancies that create a defect state that suppresses charge recombination. It was found that the conduction band (CB) of MGCN was located at a more negative position (E CB  = -0.33 V) than (O 2 /O 2 •- ) and the valence band (VB) was placed at a more positive position (E VB  = 2.30 V) than (H 2 O/OH • ), which allows generation of both radicals for photodegradation. Based on the cell viability test, the photodegraded TC in the water how non-toxicity toward Balb/c 3T3 cells after being irradiated (λ > 420 nm) for 240 min under visible light. The MGCN prepared in this study demonstrated the highest effectiveness and recyclable photocatalyst for the removal of TC among all GCNs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The extensive use of tetracycline antibiotics has led to the widespread presence of tetracycline-resistance genes in Gram-negative bacteria and this poses serious threats to human and animal health. In our previous study, we reported a method for rapid detection of Tet(X)-producers using MALDI-TOF MS. However, there have been multiple machineries involved in tetracycline resistance including efflux pump, and ribosomal protection protein. Our previous demonstrated the limitation in probing the non-Tet(X)-producing tetracycline-resistant strains. In this regard, we further developed a MALDI-TOF MS method to detect and differentiate Tet(X)-producers and non-Tet(X)-producing tetracycline-resistant strains. Test strains were incubated with tigecycline and oxytetracycline in separate tubes for 3 h and then analyzed spectral peaks of tigecycline, oxytetracycline, and their metabolite. Strains were distinguished using MS ratio for [metabolite/(metabolite+ tigecycline or oxytetracycline)]. Four control strains and 319 test strains were analyzed and the sensitivity was 98.90% and specificity was 98.34%. This was consistent with the results obtained from LC-MS/MS analysis. Interestingly, we also found that the reactive oxygen species (ROS) produced by tetracycline-susceptible strains were able to promote the degradation of oxytetracycline. Overall, the MALDI Tet(X)-plus test represents a rapid and reliable method to detect Tet(X)-producers, non-Tet(X)-producing tetracycline-resistant strains, and tetracycline-susceptible strains.

In this work, NiFe 2 O 4 /g-C 3 N 4 heterostructure was prepared and used for the photocatalytic decomposition of tetracycline hydrochloride antibiotic and for inactivation of E. coli bacteria. The fabricated NiFe 2 O 4 /g-C 3 N 4 composite displayed enhanced ability for photodegradation of organic pollutants and disinfection activities compared to the bare samples, because of the enhancement of visible light absorbance, heterojunction formation and photo-Fenton process. The optimized sample 10%-NiFe 2 O 4 /g-C 3 N 4 has photodegraded 94.5% of tetracycline hydrochloride in 80 min. The active species trapping experiments revels that ·O 2 - , h + and •OH are key decomposing species participated in the antibiotic degradation. It is hoped that the present study will provide a better understanding to fabricate efficient photocatalysts for the decomposition of organic pollutants and disinfection of bacteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Tetracycline-inducible systems are widely used control elements for mammalian gene expression. Despite multiple iterations to improve inducibility, their use is still compromised by basal promoter activity in the absence of tetracyclines. In a mammalian system, we previously showed that the introduction of the G72V mutation in the rtTA-M2 tetracycline activator lowers the basal level expression and increases the fold-induction of multiple genetic elements in a long chimeric antigen receptor construct. In this study, we confirmed that the G72V mutation was effective in minimising background expression in the absence of an inducer, resulting in an increase in fold-expression. Loss of responsiveness due to the G72V mutation was compensated through the incorporation of four sensitivity enhancing (SE) mutations, without compromising promoter tightness. However, SE mutations alone (without G72V) led to undesirable leakiness. Although cryptic splice site removal from rtTA did not alter the inducible control of the luciferase reporter gene in this simplified vector system, this is still recommended as a precaution in more complex multi-gene elements that contain rtTA. The optimized expression construct containing G72V and SE mutations currently provides the best improvement of fold-induction mediated by the rtTA-M2 activator in a mammalian system.

The present study focused on the enhancement of degradation of an important pharmaceutical pollutant, tetracycline with the help of nano photocatalyst under visible light irradiation. The study found that the synergetic effect of novel MgFe 2 O 4 -V 2 O 5 enhanced the photocatalytic degradation of tetracycline. Here, the photocatalyst was synthesized by sonochemical technique. Scanning electron microscopy image indicates the coupling of MgFe 2 O 4 nanocapsules on the surface of the V 2 O 5 nanorod. The bandgap of MgFe 2 O 4 (1.8 eV) and V 2 O 5 (2.5 eV) was shifted to 2.32 eV in MgFe 2 O 4 -V 2 O 5 to promote visible-light harvesting and it was depicted by the UV-visible DRS. XPS was used to identify the presence of chemical states with the existence of Mg 1s, Fe 2p, V 2p, and O 1s. The electrochemical impedance spectroscopy and photoluminescence spectra indicate the better separation of charge carriers owing to the formation of type II heterojunction formation. The tetracycline (25 mg/L) was degraded with MgFe 2 O 4 -V 2 O 5 (150 mg/L) that exhibited 3.3 and 5 folds enhanced rates than its counterparts (MgFe 2 O 4 and V 2 O 5 ) owing to synergism. The possible intermediate formation and degradation pathway was determined based on GC/MS analysis. TOC analysis of end products indicated maximum mineralization of tetracycline. The MgFe 2 O 4 -V 2 O 5 showed excellent recycling ability and reusability. The key photo-degradation of tetracycline was occurred by the generation of hydroxyl radicals. The MgFe 2 O 4 -V 2 O 5 exhibited high antibacterial activity that ensures the dual functionality of the prepared nanocomposites (NCs). Therefore, the present study displays MgFe 2 O 4 decorated V 2 O 5 nanorod as an ideal candidate for environmental remediation., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Staphylococcus aureus (S. aureus) infection is difficult to fight, previous experimental reports have demonstrated thioridazine (TZ) and tetracycline (TC) is an inhibitor of S. aureus efflux pump NorA and autolysin Atl, respectively, here, by means of molecular docking and molecular dynamics simulation, we observed that thioridazine (TZ) and tetracycline (TC) blocked the binding of substrates to NorA and Atl, respectively, and reduced their activities, and our antibacterial susceptibility test and three-dimensional checkerboard method showed that the three-drug combination of antibiotic cloxacillin (CXN), TZ and TC had a synergistic anti-Staphylococcal activity in vitro, and α-Hemolysin tests and scanning electron microscopy showed that the three-drug combination and the subinhibitory concentration of the combination significantly inhibited the secretion of α-hemolysin relative to the number of membrane-derived vesicles produced by S. aureus. Whereas Western blot and pharmacological inhibition assays showed that the three-drug combination significantly inhibited the expression of MAPK/NF-κB/NLRP3 proteins in macrophages induced with S. aureus α-hemolysin. In vivo, the drug combination significantly reduced bacterial colony-forming unit counts in the viscera of a mouse peritonitis model of S. aureus infection, therapy reduced the primary inflammatory pathology and the bacteria-stimulated release of cytokines such as IL-1β and TNF-α, and inhibited the expression of MAPK/NF-κB/NLRP3 proteins in peritoneal macrophages. Thus, the combination of efflux pump inhibitor, autolysis inhibitor and antibiotic, is a novel anti-Staphylococcal and anti-inflammatory strategy who owning good antibacterial activity and significant inhibiting staphylococcal α-hemolysin and inflammation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Combining multiple drugs or biologically active substances for wound healing could not only resist the formation of multidrug resistant pathogens, but also achieve better therapeutic effects. Herein, the hydrophobic fluoroquinolone antibiotic ciprofloxacin (CIP) and the hydrophilic broad-spectrum antibiotic tetracycline hydrochloride (TH) were introduced into the coaxial polycaprolactone/gelatin (PCL/GEL) nanofiber mat with CIP loaded into the PCL (core layer) and TH loaded into the GEL (shell layer), developing antibacterial wound dressing with the co-delivering of the two antibiotics (PCL-CIP/GEL-TH). The nanostructure, physical properties, drug release, antibacterial property, and in vitro cytotoxicity were investigated accordingly. The results revealed that the CIP shows a long-lasting release of five days, reaching the releasing rate of 80.71%, while the cumulative drug release of TH reached 83.51% with a rapid release behavior of 12 h. The in vitro antibacterial activity demonstrated that the coaxial nanofiber mesh possesses strong antibacterial activity against E. coli and S. aureus . In addition, the coaxial mats showed superior biocompatibility toward human skin fibroblast cells (hSFCs). This study indicates that the developed PCL-CIP/GEL-TH nanofiber membranes hold enormous potential as wound dressing materials.

The effect of tetracycline (TC) on nitrogen removal in wastewater treatment plants has become a new problem. This study investigated the effects of TC on nitrogen removal using a Moving Bed Biofilm Reactor system. The results showed that there was no significant effect on nitrogen removal performance when the concentration of TC was 5 mg/L, and that the total nitrogen (TN) removal efficiency could reach 75-77%. However, when the concentration of TC increased to 10 mg/L, the denitrification performance was affected and the TN removal efficiency decreased to 58%. The abundance of denitrifying bacteria such as those in the genus Thauera decreased, and TC-resistant bacteria gradually became dominant. At a TC concentration of 10 mg/L, there were also increases and decreases, respectively, in the abundance of resistance and denitrification functional genes. The inhibitory effect of TC on denitrification was achieved mainly by the inhibition of nitrite-reducing bacteria., Competing Interests: The authors have declared that no competing interests exist.