Selective increase of antibiotic-resistant denitrifiers drives N2O production in ciprofloxacin-contaminated soils.

Agricultural systems significantly contribute to global N2O emissions, which is intensified by excessive fertilization and antibiotic residues, attracting global concerns. However, the dynamics and pathways of antibiotics-induced soil N2O production coupled with microbial metabolism remain controversial. Here, we explored the pathways of N2O production in agricultural soils exposed to ciprofloxacin (CIP), and revealed the underlying mechanisms of CIP degradation and the associated microbial metabolisms using 15N-isotope labeling and molecular techniques. CIP exposure significantly increases the total soil N2O production rate. This is attributed to an unexpected shift from heterotrophic and autotrophic nitrification to denitrification and an increased abundance of denitrifiers Methylobacillus members under CIP exposure. The most striking strain M. flagellatus KT is further discovered to harbor N2O-producing genes but lacks a N2O-reducing gene, thereby stimulating denitrification-based N2O production. Moreover, this denitrifying strain is probably capable of utilizing the byproducts of CIP as carbon sources, evidenced by genes associated with CIP resistance and degradation. Molecular docking further shows that CIP is well ordered in the catalytic active site of CotA laccase, thus affirming the potential for this strain to degrade CIP. These findings advance the mechanistic insights into N2O production within terrestrial ecosystems coupled with the organic contaminants degradation.

Molecular Mechanism of Ciprofloxacin Translocation Through the Major Diffusion Channels of the ESKAPE Pathogens Klebsiella pneumoniae and Enterobacter cloacae.

Experimental studies on the translocation and accumulation of antibiotics in Gram-negative bacteria have revealed details of the properties that allow efficient permeation through bacterial outer membrane porins. Among the major outer membrane diffusion channels, OmpF has been extensively studied to understand the antibiotic translocation process. In a few cases, this knowledge has also helped to improve the efficacy of existing antibacterial molecules. However, the extension of these strategies to enhance the efficacy of other existing and novel drugs require comprehensive molecular insight into the permeation process and an understanding of how antibiotic and channel properties influence the effective permeation rates. Previous studies have investigated how differences in antibiotic charge distribution can influence the observed permeation pathways through the OmpF channel, and have shown that the dynamics of the L3 loop can play a dominant role in the permeation process. Here, we perform all-atom simulations of the OmpF orthologs, OmpE35 from Enterobacter cloacae and OmpK35 from Klebsiella pneumoniae. Unbiased simulations of the porins and biased simulations of the ciprofloxacin permeation processes through these channels provide insight into the differences in the permeation pathway and energetics. In addition, we show that similar to the OmpF channel, antibiotic-induced dynamics of the L3 loop are also operative in the orthologs. However, the sequence and structural differences, influence the extent of the L3 loop fluctuations with OmpK35 showing greater stability in unbiased runs and subdued fluctuations in simulations with ciprofloxacin.

Drug metabolism of ciprofloxacin, ivacaftor, and raloxifene by Pseudomonas aeruginosa cytochrome P450 CYP107S1.

Drug metabolism is one of the main processes governing the pharmacokinetics and toxicity of drugs via their chemical biotransformation and elimination. In humans, the liver, enriched with cytochrome P450 (CYP) enzymes, plays a major metabolic and detoxification role. The gut microbiome and its complex community of microorganisms can also contribute to some extent to drug metabolism. However, during an infection when pathogenic microorganisms invade the host, our knowledge of the impact on drug metabolism by this pathobiome remains limited. The intrinsic resistance mechanisms and rapid metabolic adaptation to new environments often allow the human bacterial pathogens to persist, despite the many antibiotic therapies available. Here, we demonstrate that a bacterial CYP enzyme, CYP107S1, from Pseudomonas aeruginosa, a predominant bacterial pathogen in cystic fibrosis patients, can metabolize multiple drugs from different classes. CYP107S1 demonstrated high substrate promiscuity and allosteric properties much like human hepatic CYP3A4. Our findings demonstrated binding and metabolism by the recombinant CYP107S1 of fluoroquinolone antibiotics (ciprofloxacin and fleroxacin), a cystic fibrosis transmembrane conductance regulator potentiator (ivacaftor), and a selective estrogen receptor modulator antimicrobial adjuvant (raloxifene). Our in vitro metabolism data were further corroborated by molecular docking of each drug to the heme active site using a CYP107S1 homology model. Our findings raise the potential for microbial pathogens modulating drug concentrations locally at the site of infection, if not systemically, via CYP-mediated biotransformation reactions. To our knowledge, this is the first report of a CYP enzyme from a known bacterial pathogen that is capable of metabolizing clinically utilized drugs.

Ciprofloxacin affects nutrient removal in manganese ore-based constructed wetlands: Adaptive responses of macrophytes and microbes.

Ciprofloxacin (CIP) has received considerable attention in recent decades due to its high ecological risk. However, little is known about the potential response of macrophytes and microbes to varying levels of CIP exposure in constructed wetlands. Therefore, lab-scale manganese ore-based tidal flow constructed wetlands (MO-TFCWs) were operated to evaluate the responses of macrophytes and microbes to CIP over the long term. The results indicated that total nitrogen removal improved from 79.93% to 87.06% as CIP rose from 0 to 4 mg L-1. The chlorophyll content and antioxidant enzyme activities in macrophytes were enhanced under CIP exposure, but plant growth was not inhibited. Importantly, CIP exposure caused a marked evolution of the substrate microbial community, with increased microbial diversity, expanded niche breadth and enhanced cooperation among the top 50 genera, compared to the control (no CIP). Co-occurrence network also indicated that microorganisms may be more inclined to co-operate than compete. The abundance of the keystone bacterium (involved in nitrogen transformation) norank_f__A0839 increased from 0.746% to 3.405%. The null model revealed drift processes (83.33%) dominated the community assembly with no CIP and 4 mg L-1 CIP. Functional predictions indicated that microbial carbon metabolism, electron transfer and ATP metabolism activities were enhanced under prolonged CIP exposure, which may contribute to nitrogen removal. This study provides valuable insights that will help achieve stable nitrogen removal from wastewater containing antibiotic in MO-TFCWs.

Biodegradation of ciprofloxacin using machine learning tools: Kinetics and modelling.

Recently, the rampant administration of antibiotics and their synthetic organic constitutes have exacerbated adverse effects on ecosystems, affecting the health of animals, plants, and humans by promoting the emergence of extreme multidrug-resistant bacteria (XDR), antibiotic resistance bacterial variants (ARB), and genes (ARGs). The constraints, such as high costs, by-product formation, etc., associated with the physico-chemical treatment process limit their efficacy in achieving efficient wastewater remediation. Biodegradation is a cost-effective, energy-saving, sustainable alternative for removing emerging organic pollutants from environmental matrices. In view of the same, the current study aims to explore the biodegradation of ciprofloxacin using microbial consortia via metabolic pathways. The optimal parameters for biodegradation were assessed by employing machine learning tools, viz. Artificial Neural Network (ANN) and statistical optimization tool (Response Surface Methodology, RSM) using the Box-Behnken design (BBD). Under optimal culture conditions, the designed bacterial consortia degraded ciprofloxacin with 95.5% efficiency, aligning with model prediction results, i.e., 95.20% (RSM) and 94.53% (ANN), respectively. Thus, befitting amendments to the biodegradation process can augment efficiency and lead to a greener solution for antibiotic degradation from aqueous media.

A new understanding on the prerequisite of antibiotic biodegradation in wastewater treatment: Adhesive behavior between antibiotic-degrading bacteria and ciprofloxacin.

The extensive exploration of antibiotic biodegradation by antibiotic-degrading bacteria in biological wastewater treatment processes has left a notable gap in understanding the behavior of these bacteria when exposed to antibiotics and the initiation of biodegradation processes. This study, therefore, delves into the adhesive behavior of Paraclostridium bifermentans, isolated from a bioreactor treating ciprofloxacin-laden wastewater, towards ciprofloxacin molecules. For the first time, this behavior is observed and characterized through quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy. The investigation further extends to identify key regulatory factors and mechanisms governing this adhesive behavior through a comparative proteomics analysis. The results reveal the dominance of extracellular proteins, particularly those involved in nucleotide binding, hydrolase, and transferase, in the adhesion process. These proteins play pivotal roles through direct chemical binding and the regulation of signaling molecule. Furthermore, QCM-D measurements provide evidence that transferase-related signaling molecules, especially tyrosine, augment the binding between ciprofloxacin and transferases, resulting in enhance ciprofloxacin removal by P. bifermentans (increased by ∼1.2-fold). This suggests a role for transferase-related signaling molecules in manipulating the adhesive behavior of P. bifermentans towards ciprofloxacin. These findings contribute to a new understanding of the prerequisites for antibiotic biodegradation and offer potential strategies for improving the application of antibiotic-degrading bacteria in the treatment of antibiotics-laden wastewater.

Removal efficiencies of seven frequently detected antibiotics and related physiological responses in three microalgae species.

The main objective of this study is to investigate the effect of mixtures of seven widely used human antibiotics (ciprofloxacin, clarithromycin, erythromycin, metronidazole, ofloxacin, sulfamethoxazole, and trimethoprim) on the growth, pH, pigment production, and antibiotics removal of three microalgal species (Auxenochlorella protothecoides, Tetradesmus obliquus, and Chlamydomonas acidophila). Batch assays were conducted with media with antibiotic mixtures at 10, 50, and 100 µg L-1 for each antibiotic. The three microalgae species effectively removed the antibiotics without any growth inhibition, even when exposed to the highest antibiotic concentrations. Biosorption was reported as the primary mechanism for ciprofloxacin, clarithromycin, metronidazole, and ofloxacin, with up to 70% removal, especially in A. protothecoides and C. acidophila. A. protothecoides, a species never investigated for antibiotic removal, was the only microalgae exhibiting bioaccumulation and biodegradation of specific antibiotics, including sulfamethoxazole. Furthermore, in media with the highest antibiotic concentration, all three species exhibited increased chlorophyll (up to 37%) and carotenoid (up to 32%) production, accompanied by a pH decrease of 3 units. Generally, in the present study, it has been observed that physiological responses and the removal of antibiotics by microalgae are interlinked and contingent on the antibiotic levels and types.

Physiological responses and removal mechanisms of ciprofloxacin in freshwater microalgae.

Antibiotics, such as ciprofloxacin (CIP), are frequently detected in various environmental compartments, posing significant risks to ecosystems and human health. In this study, the physiological responses and elimination mechanisms of CIP in Chlorella sorokiniana and Scenedesmus dimorphus were determined. The exposure CIP had a minimal impact on the growth of microalgae, with maximum inhibit efficiency (IR) of 5.14% and 22.74 for C. sorokiniana and S. dimorphus, respectively. Notably, the photorespiration in S. dimorphus were enhanced. Both microalgae exhibited efficient CIP removal, predominantly through bioaccumulation and biodegradation processes. Intermediates involved in photolysis and biodegradation were analyzed through Liquid Chromatography High Resolution Mass Spectrometer (HPLC-MS/MS), providing insights into degradation pathways of CIP. Upregulation of key enzymes, such as dioxygenase, oxygenase and cytochrome P450, indicated their involvement in the biodegradation of CIP. These findings enhance our understanding of the physiological responses, removal mechanisms, and pathways of CIP in microalgae, facilitating the advancement of microalgae-based wastewater treatment approaches, particularly in antibiotic-contaminated environments.

Insights into the molecular network underlying phytotoxicity and phytoaccumulation of ciprofloxacin.

Ciprofloxacin (CIP) is frequently detected in agricultural soils and can be accumulated by crops, causing phytotoxicities and food safety concerns. However, the molecular basis of its phytotoxicity and phytoaccumulation is hardly known. Here, we analyzed physiological and molecular responses of choysum (Brassica parachinensis) to CIP stress by comparing low CIP accumulation variety (LAV) and high accumulation variety (HAV). Results showed that the LAV suffered more severe inhibition of growth and photosynthesis than the HAV, exhibiting a lower tolerance to CIP toxicity. Integrated transcriptome and proteome analyses suggested that more differentially expressed genes/proteins (DEGs/DEPs) involved in basic metabolic processes were downregulated to a larger extent in the LAV, explaining its lower CIP tolerance at molecular level. By contrast, more DEGs/DEPs involved in defense responses were upregulated to a larger extent in the HAV, showing the molecular basis of its stronger CIP tolerance. Further, a CIP phytotoxicity-responsive molecular network was constructed for the two varieties to better understand the molecular mechanisms underlying the variety-specific CIP tolerance and accumulation. The results present the first comprehensive molecular profile of plant response to CIP stress for molecular-assisted breeding to improve CIP tolerance and minimize CIP accumulation in crops.

Insight into the on/off switch that regulates expression of the MSMEG-3762/63 efflux pump in Mycobacterium smegmatis.

Drug resistance is one of the most difficult challenges facing tuberculosis (TB) control. Drug efflux is among the mechanisms leading to drug resistance. In our previous studies, we partially characterized the ABC-type MSMEG-3762/63 efflux pump in Mycobacterium smegmatis, which shares high percentage of identity with the Mycobacterium tuberculosis Rv1687/86c pump. MSMEG-3762/63 was shown to have extrusion activity for rifampicin and ciprofloxacin, used in first and second-line anti-TB treatments. Moreover, we described the functional role of the TetR-like MSMEG-3765 protein as a repressor of the MSMEG_3762/63/65 operon and orthologous Rv1687/86/85c in M. tuberculosis. Here we show that the operon is upregulated in the macrophage environment, supporting a previous observation of induction triggered by acid-nitrosative stress. Expression of the efflux pump was also induced by sub-inhibitory concentrations of rifampicin or ciprofloxacin. Both these drugs also prevented the binding of the MSMEG-3765 TetR repressor protein to its operator in the MSMEG_3762/63/65 operon. The hypothesis that these two drugs might be responsible for the induction of the efflux pump operon was assessed by bioinformatics analyses. Docking studies using a structural model of the regulator MSMEG-3765 showed that both antibiotics abolished the ability of this transcriptional repressor to recognize the efflux pump operon by interacting with the homodimer at different binding sites within the same binding pocket. Reduced binding of the repressor leads to induction of the efflux pump in M. smegmatis, and reduced efficacy of these two anti-mycobacterial drugs.

Neurobehavioral impairments in ciprofloxacin- treated osteoarthritic adult rats.

Background and purpose:

Ciprofloxacin (CIP) is a broad-spectrum antibiotic widely used in clinical practice to treat musculoskeletal infections. Fluoroquinolone-induced neurotoxic adverse events have been reported in a few case reports, all the preclinical studies on its neuropsychiatric side effects involved only healthy animals. This study firstly investigated the behavioral effects of CIP in an osteoarthritis rat model with joint destruction and pain, which can simulate inflammation-associated musculoskeletal pain. Furthermore, effects of CIP on regional brain-derived neurotrophic factor (BDNF) expression were examined given its major contributions to the neuromodulation and plasticity underlying behavior and cognition. 

Fourteen days after induction of chronic osteoarthritis, animals were administered vehicle, 33 mg/kg or 100 mg/kg CIP for five days intraperitoneally. Motor activity, behavioral motivation, and psychomotor learning were examined in a reward-based behavioral test (Ambitus) on Day 4 and sensorimotor gating by the prepulse inhibition test on Day 5. Thereafter, the prolonged BDNF mRNA and protein expression levels were measured in the hippocampus and the prefrontal cortex. 

CIP dose-dependently reduced both locomotion and reward-motivated exploratory activity, accompanied with impaired learning ability. In contrast, there were no significant differences in startle reflex and sensory gating among treatment groups; however, CIP treatment reduced motor activity of the animals in this test, too. These alterations were associated with reduced BDNF mRNA and protein expression levels in the hippocampus but not the prefrontal cortex. 

This study revealed the detrimental effects of CIP treatment on locomotor activity and motivation/learning ability during osteoarthritic condition, which might be due to, at least partially, deficient hippocampal BDNF expression and ensuing impairments in neural and synaptic plasticity.

Nutrient condition modulates the antibiotic tolerance of Pseudomonas aeruginosa.

The variation in nutrient content across diverse environments has a significant impact on the survival and metabolism of microorganisms. In this study, we examined the influence of nutrients on the antibiotic tolerance of the PAO1 strain of Pseudomonas aeruginosa. Our findings indicate that under nutrient-rich conditions, this strain exhibited relatively high tolerance to ceftazidime, chloramphenicol, and tetracycline, but not aminoglycosides and fluoroquinolones. Transcriptome analysis revealed that genes associated with antibiotic tolerance were expressed more efficiently in nutrient-rich media, including ribosomal protein genes and multidrug efflux pump genes, which conferred higher tetracycline tolerance to the strain. Furthermore, the genes responsible for translation, biosynthesis, and oxidative phosphorylation were suppressed when nutrients were limited, resulting in decreased metabolic activity and lower sensitivity to ciprofloxacin. Artificial interference with ATP synthesis utilizing arsenate confirmed that the curtailment of energy provision bolstered the observed tolerance to ciprofloxacin. In general, our results indicate that this strain of P. aeruginosa tends to activate its intrinsic resistance mechanisms in nutrient-rich environments, thereby enhancing resistance to certain antibiotics. Conversely, in nutrient-limited environments, the strain is more likely to enter a dormant state, which enables it to tolerate antibiotics to which it would otherwise be sensitive. These findings further suggest that antibiotics released in environments with varying eutrophication levels may have divergent effects on the development of bacterial antibiotic resistance.

An integrated quorum quenching biocatalytic nanoplatform for synergistic chemo-photothermal eradication of P. aeruginosa biofilm infections.

Decontamination of biofilm-associated infections presents a significant challenge due to the physical and chemical barrier created by the formation of extracellular matrices. This barrier restricts the access of antibiotics to the bacterial communities within the biofilm and provides protection to the persister cells, potentially leading to antibiotic resistance. In this study, we have developed an integrated quorum quenching biocatalytic nanoplatform for the synergistic chemo-photothermal eradication of P. aeruginosa biofilm infections. Ciprofloxacin (Cip), a model antibiotic, was absorbed onto PDA NPs through π-π stacking. Additionally, acylase (AC) was immobilized on PDA NPs through Schiff base reaction and Michael addition, resulting in the formation of the biocatalytic nanoplatform (PDA-Cip-AC NPs). This biocatalytic nanoplatform was able to enzymatically degrade AHL signaling molecules, thus achieving efficient quorum quenching activity to prevent biofilm formation. Furthermore, the NIR light-triggered on-demand Ciprofloxacin release further enhanced the eradication of P. aeruginosa biofilm infections with a synergy of local hyperthermia. We envision that this integrated quorum quenching nanoplatform provides a reliable tool for combating P. aeruginosa biofilm infections. STATEMENT OF SIGNIFICANCE: An integrated quorum quenching biocatalytic nanoplatform has been developed for the eradication of P. aeruginosa biofilm infections. Quorum-sensing signals play a crucial role in modulating bacterial cell-to-cell communication, biofilm formation, and secretion of virulence factors. This biocatalytic nanoplatform efficiently degrades AHL signaling molecules, thereby blocking cell-to-cell communication and preventing biofilm formation. Additionally, local hyperthermia and on-demand Ciprofloxacin release were achieved through NIR irradiation, working synergistically to eradicate P. aeruginosa biofilm infections.

DinB (DNA polymerase IV), ImuBC and RpoS contribute to the generation of ciprofloxacin-resistance mutations in Pseudomonas aeruginosa.

We investigated the role(s) of the damage-inducible SOS response dinB and imuBC gene products in the generation of ciprofloxacin-resistance mutations in the important human opportunistic bacterial pathogen, Pseudomonas aeruginosa. We found that the overall numbers of ciprofloxacin resistant (CipR) mutants able to be recovered under conditions of selection were significantly reduced when the bacterial cells concerned carried a defective dinB gene, but could be elevated to levels approaching wild-type when these cells were supplied with the dinB gene on a plasmid vector; in turn, firmly establishing a role for the dinB gene product, error-prone DNA polymerase IV, in the generation of CipR mutations in P. aeruginosa. Further, we report that products of the SOS-regulated imuABC gene cassette of this organism, ImuB and the error-prone ImuC DNA polymerase, are also involved in generating CipR mutations in this organism, since the yields of CipR mutations were substantially decreased in imuB- or imuC-defective cells compared to wild-type. Intriguingly, we found that the mutability of a dinB-defective strain could not be rescued by overexpression of the imuBC genes. And similarly, overexpression of the dinB gene either only modestly or else failed to restore CipR mutations in imuB- or imuC-defective cells, respectively. Combined, these results indicated that the products of the dinB and imuBC genes were acting in the same pathway leading to the generation of CipR mutations in P. aeruginosa. In addition, we provide evidence indicating that the general stress response sigma factor σs, RpoS, is required for mutagenesis in this organism and is in part at least modulating the dinB (DNA polymerase IV)-dependent mutational process. Altogether, these data provide further insight into the complexity and multifaceted control of the mutational mechanism(s) contributing to the generation of ciprofloxacin-resistance mutations in P. aeruginosa.

Comparative study of the toxicity mechanisms of quinolone antibiotics on soybean seedlings: Insights from molecular docking and transcriptomic analysis.

The ecological effects of quinolone antibiotics (QNs) on non-target organisms have received widespread attention. The toxicological mechanisms of three common QNs, that is, enrofloxacin, levofloxacin, and ciprofloxacin, on soybean seedlings were investigated in this study. Enrofloxacin and levofloxacin caused significant growth inhibition, ultrastructural alterations, photosynthetic suppression, and stimulation of the antioxidant system, with levofloxacin exhibiting the strongest toxic effects. Ciprofloxacin (<1 mg·L-1) did not have a significant effect on the soybean seedlings. As the concentrations of enrofloxacin and levofloxacin increased, antioxidant enzyme activities, malondialdehyde content, and hydrogen peroxide levels also increased. Meanwhile, the chlorophyll content and chlorophyll fluorescence parameters decreased, indicating that the plants underwent oxidative stress and photosynthesis was suppressed. The cellular ultrastructure was also disrupted, which was manifested by swollen chloroplasts, increased starch granules, disintegration of plastoglobules, and mitochondrial degradation. The molecular docking results suggested that the QNs have an affinity for soybean target protein receptors (4TOP, 2IUJ, and 1FHF), with levofloxacin having the highest binding energy (-4.97, -3.08, -3.8, respectively). Transcriptomic analysis has shown that genes were upregulated under the enrofloxacin and levofloxacin treatments were mainly involved in ribosome metabolism and processes to synthesize oxidative stress-related proteins. Downregulated genes in the levofloxacin treatment were primarily enriched in photosynthesis-related pathways, indicating that levofloxacin significantly inhibited gene expression for photosynthesis. Genes expression level by quantitative real-time PCR analysis was consistent with the transcriptomic results. This study confirmed the toxic effect of QNs on soybean seedlings, and provided new insights into the environmental risks of antibiotics.

Bromotyrosine-Derived Metabolites from a Marine Sponge Inhibit Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa forms stable biofilms, providing a major barrier for multiple classes of antibiotics and severely impairing treatment of infected patients. The biofilm matrix of this Gram-negative bacterium is primarily composed of three major exopolysaccharides: alginate, Psl, and Pel. Here, we studied the antibiofilm properties of sponge-derived natural products ianthelliformisamines A-C and their combinations with clinically used antibiotics. Wild-type P. aeruginosa strain and its isogenic exopolysaccharide-deficient mutants were employed to determine the interference of the compounds with biofilm matrix components. We identified that ianthelliformisamines A and B worked synergistically with ciprofloxacin to kill planktonic and biofilm cells. Ianthelliformisamines A and B reduced the minimum inhibitory concentration (MIC) of ciprofloxacin to 1/3 and 1/4 MICs, respectively. In contrast, ianthelliformisamine C (MIC = 53.1 µg/mL) alone exhibited bactericidal effects dose-dependently on both free-living and biofilm populations of wild-type PAO1, PAO1ΔpslA (Psl deficient), PDO300 (alginate overproducing and mimicking clinical isolates), and PDO300Δalg8 (alginate deficient). Interestingly, the biofilm of the clinically relevant mucoid variant PDO300 was more susceptible to ianthelliformisamine C than strains with impaired polysaccharide synthesis. Ianthelliformisamines exhibited low cytotoxicity towards HEK293 cells in the resazurin viability assay. Mechanism of action studies showed that ianthelliformisamine C inhibited the efflux pump of P. aeruginosa. Metabolic stability analyses indicated that ianthelliformisamine C is stable and ianthelliformisamines A and B are rapidly degraded. Overall, these findings suggest that the ianthelliformisamine chemotype could be a promising candidate for the treatment of P. aeruginosa biofilms.

Ciprofloxacin enhances cadmium toxicity to earthworm Eisenia fetida by altering the gut microorganism composition.

The concurrent existence of cadmium (Cd) and ciprofloxacin (CIP) in agricultural soils is very common, but presents a challenge to soil organisms. As more attention has been paid to the effect of toxic metals on the migration of antibiotic resistance genes, the critical role of the gut microbiota in CIP-modifying Cd toxicity in earthworms remains unclear. In this study, Eisenia fetida was exposed to Cd and CIP alone or in combination at environmentally relevant concentrations. Cd and CIP accumulation in earthworm increased as their respective spiked concentrations increased. In fact, Cd accumulation increased by 39.7% when 1 mg/kg CIP was added; however, the addition of Cd did not affect CIP uptake. Compared with exposure to Cd alone, a greater ingestion of Cd following combined exposure to Cd and 1 mg/kg CIP resulted in greater oxidative stress and energy metabolism disturbances in earthworms. The reactive oxygen species (ROS) contents and apoptosis rate of coelomocytes were more sensitive to Cd than these biochemical indicators. In fact, 1 mg/kg Cd induced the derivation of ROS. Similarly, the toxicity of Cd (5 mg/kg) to coelomocytes was promoted by CIP (1 mg/kg), ROS content in coelomocytes and the apoptosis rate increased by 29.2% and 113.1%, respectively, due to increased Cd accumulation. Further investigation of the gut microorganisms revealed that the decreased abundance of Streptomyces strains (known as Cd accumulation taxa) could be a critical factor for enhanced Cd accumulation and greater Cd toxicity to earthworms following exposure to both Cd and CIP; this was because this microorganism group was eliminated by the simultaneous ingestion of CIP. This study stressed the role of gut microorganisms in altering the toxicity of Cd and CIP combined contamination in soil organisms. More attention should be paid to the ecological risks of such combined contamination in soils.

An rRNA fragment in extracellular vesicles secreted by human airway epithelial cells increases the fluoroquinolone sensitivity of P. aeruginosa.

Lung infections caused by antibiotic-resistant strains of Pseudomonas aeruginosa are difficult to eradicate in immunocompromised hosts such as those with cystic fibrosis. We previously demonstrated that extracellular vesicles (EVs) secreted by primary human airway epithelial cells (AECs) deliver microRNA let-7b-5p to P. aeruginosa to suppress biofilm formation and increase sensitivity to beta-lactam antibiotics. In this study, we show that EVs secreted by AECs transfer multiple distinct short RNA fragments to P. aeruginosa that are predicted to target the three subunits of the fluoroquinolone efflux pump MexHI-OpmD, thus increasing antibiotic sensitivity. Exposure of P. aeruginosa to EVs resulted in a significant reduction in the protein levels of MexH (-48%), MexI (-50%), and OpmD (-35%). Moreover, EVs reduced planktonic growth of P. aeruginosa in the presence of the fluoroquinolone antibiotic ciprofloxacin by 20%. A mexGHI-opmD deletion mutant of P. aeruginosa phenocopied this increased sensitivity to ciprofloxacin. Finally, we found that a fragment of an 18S ribosomal RNA (rRNA) external transcribed spacer that was transferred to P. aeruginosa by EVs reduced planktonic growth of P. aeruginosa in the presence of ciprofloxacin, reduced the minimum inhibitory concentration of P. aeruginosa for ciprofloxacin by over 50%, and significantly reduced protein levels of both MexH and OpmD. In conclusion, an rRNA fragment secreted by AECs in EVs that targets the fluoroquinolone efflux pump MexHI-OpmD downregulated these proteins and increased the ciprofloxacin sensitivity of P. aeruginosa. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with ciprofloxacin-resistant P. aeruginosa infections.NEW & NOTEWORTHY Human RNA fragments transported in extracellular vesicles interfere with Pseudomonas aeruginosa drug efflux pumps. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections.

Effects of the order of exposure to antimicrobials on the incidence of multidrug-resistant Pseudomonas aeruginosa.

Multidrug-resistant Pseudomonas aeruginosa (MDRP) is one of the most important pathogens in clinical practice. To clarify the mechanisms contributing to its emergence, we isolated MDRPs using the P. aeruginosa PAO1, the whole genome sequence of which has already been elucidated. Mutant strains resistant to carbapenems, aminoglycosides, and new quinolones, which are used to treat P. aeruginosa infections, were isolated; however, none met the criteria for MDRPs. Then, PAO1 strains were exposed to these antimicrobial agents in various orders and the appearance rate of MDRP varied depending on the order of exposure; MDRPs more frequently appeared when gentamicin was applied before ciprofloxacin, but were rarely isolated when ciprofloxacin was applied first. Exposure to ciprofloxacin followed by gentamicin increased the expression of MexCD-OprJ, an RND-type multidrug efflux pump, due to the NfxB mutation. In contrast, exposure to gentamicin followed by ciprofloxacin resulted in more mutations in DNA gyrase. These results suggest that the type of quinolone resistance mechanism is related to the frequency of MDRP and that the risk of MDRP incidence is highly dependent on the order of exposure to gentamicin and ciprofloxacin.

XerC Is Required for the Repair of Antibiotic- and Immune-Mediated DNA Damage in Staphylococcus aureus.

To survive in the host environment, pathogenic bacteria need to be able to repair DNA damage caused by both antibiotics and the immune system. The SOS response is a key bacterial pathway to repair DNA double-strand breaks and may therefore be a good target for novel therapeutics to sensitize bacteria to antibiotics and the immune response. However, the genes required for the SOS response in Staphylococcus aureus have not been fully established. Therefore, we carried out a screen of mutants involved in various DNA repair pathways to understand which were required for induction of the SOS response. This led to the identification of 16 genes that may play a role in SOS response induction and, of these, 3 that affected the susceptibility of S. aureus to ciprofloxacin. Further characterization revealed that, in addition to ciprofloxacin, loss of the tyrosine recombinase XerC increased the susceptibility of S. aureus to various classes of antibiotics, as well as to host immune defenses. Therefore, the inhibition of XerC may be a viable therapeutic approach to sensitize S. aureus to both antibiotics and the immune response.