A novel enrofloxacin-degrading fungus, Humicola sp. KC0924g, isolated from the rhizosphere sediment of the submerged macrophyte Vallisneria spiralis L.

doi:10.21203/rs.3.rs-3404764/v1

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Research Article

A novel enrofloxacin-degrading fungus, Humicola sp. KC0924g, isolated from the rhizosphere sediment of the submerged macrophyte Vallisneria spiralis L.

https://doi.org/10.21203/rs.3.rs-3404764/v1

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published 20 Mar, 2024

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Herein, a novel enrofloxacin-degrading fungus was isolated from a rhizosphere sediment of the submerged macrophyte Vallisneria spiralis L.. The isolate, designated KC0924g, was identified as a member of the genus Humicola based on morphological characteristics and tandem conserved sequences analysis. The optimal temperature and pH for enrofloxacin degradation by strain KC0924g was 28°C and 9.0, respectively. Under such condition, 98.2% of enrofloxacin with initial concentration being 1 mg L^-1 was degraded after 72 h of incubation, with nine possible degradation products identified. Accordingly, four different metabolic pathways were proposed, which were initiated by cleavage of the piperazine moiety, hydroxylation of the aromatic ring, oxidative decarboxylation, or defluorination. In addition to enrofloxacin, strain KC0924g also degraded other fluoroquinolone antibiotics (ciprofloxacin, norfloxacin, and ofloxacin), malachite green (an illegal addition in aquaculture) and leucomalachite green. Pretreatment of cells of strain KC0924g with Cu²⁺ accelerated ENR degradation. Moreover, it was speculated that one flavin-dependent monooxygenase and/or one laccase involved in ENR degradation based on the increased transcriptional levels of these two genes after Cu²⁺ induction. This work enriches strain resources for enrofloxacin remediation and, more importantly, would facilitate studies on the molecular mechanism of ENR degradation with degradation-related transcriptome available.

Enrofloxacin

Biodegradation

Humicola sp.

Degradation pathway

Comparative transcriptome analysis

The output of aquatic products in China ranks at the top worldwide, mainly depending on intensive aquaculture(Han et al. 2020). This type of aquaculture has led to growing problems with microbial diseases, which now require the intensive use of antibiotics. Enrofloxacin (ENR), one type of fluoroquinolone antibiotic (FQs), has been widely used as the feed additive for disease prevention and growth promotion in aquaculture (Ma et al. 2019). However, up to 70% of the ENR was excreted in an unaltered form due to the limitation of absorption and metabolism by aquatic animals, which leads to the frequent detection of residual ENR in the environment (Van Doorslaer et al. 2014). For example, the detection frequency of ENR in samples from Qingshitan Reservoir (South China) was 100%, with the concentrations of ENR ranging from 4.59–6.06 ng L^− 1 (in water), 4.70-331.82 µg kg^− 1 (in sediment) and 6.73-102.87 µg kg^− 1 (in fish muscles). ENR residues have adverse effects on ecosystem functions and introduced the risk of spreadingantibiotic-resistant pathogens, therefore raising public concerns (Riaz et al. 2018, Bhatt and Chatterjee 2022). Thus, removing this antibiotic from the environment is urgently needed.

Physical, chemical and bioremediation methods have been adopted to remove ENR from the environment (Bhatt and Chatterjee 2022). These include adsorption by special materials, photolysis, redox reactions, electrochemical methods, phytoremediation and microbial degradation (Zhang and Huang 2007, Li et al. 2011, Moreira et al. 2020). Among them, bioremediation, such as phytoremediation and microbial degradation, is considered the most promising method due to the economic advantages, high efficiency, no secondary pollution and wide applicability. Phytoremediation is a process that uses a variety of plants and their associated rhizosphere microbes to degrade, extract, control or immobilize pollutants in the environment(Hoang et al. 2013). Currently, the use of terrestrial and aquatic plants (e.g., Glycine max L., Lactuca sativa L., Hordeum vulgare L., Cucumis sativa L., Acrostichum aureum L., Rhizophora apiculata B1. and Vallisneria spiralis L.) for FQs removal has been reported (Boonsaner and Hawker 2010, Ashiq et al. 2021, Abd El-Monaem et al. 2022). In accordance with previous studies, planting submerged plants (Vallisneria spiralis L.) for in situ remediation of ENR-contaminated sediment originated from aquaculture ponds has been proven feasible by our team (Zhang et al., 2019), with ENR removal efficiencies ranges from 32.3–74.7%. Furthermore, it was referred that ENR removal occurred via plant absorption, photodegradation and biodegradation by rhizosphere microorganisms(Zhang et al. 2019). Further research is needed to better understand the removal mechanism of ENR in sediments to better optimize this bioremediation strategy. Microorganisms are considered to be the most suitable entities for bioremediation because of their physiological abilities and metabolic capacities to degrade or detoxify pollutants. It was evidenced that the brown rot fungus Gleophyllum striatum was shown to degrade ENR via extracellular Fenton-type reaction (Wetzstein et al. 1997, Karl et al. 2006). Meanwhile, it was reported that microbial communities in the root sediments of estuarine plants can degrade ENR (Alexandrino et al. 2017, Santos et al. 2019). However, there are very few reports on ENR-degrading pure cultures isolated from the rhizosphere of aquatic plants. As such, isolation and characterization of ENR-degrading strains from this niche could provide strain resources for better application of phytoremediation (e.g., bioaugmentation in the rhizosphere). In addition, it can also provide some clues for a better understanding of the remediation mechanism. The development of microbial genomics or transcriptomics technologies provides new insight into the current understanding of xenobiotic degradation and may lead to new strategies for improving antibiotic degradation (Zhang et al. 2023). For FQs biodegradation, most previous studies have attempted to reveal the mechanisms of biodegradation in the absence of genomic or transcriptomic information.

The aims of this study were three-fold: (1) to isolate an ENR degrader from the rhizosphere sediment of Vallisneria spiralis L. which was already proved to mediate ENR remendiation, (2) to investigate the degradation performance of this strain, such as degradation efficiency, key intermediates identification together with intermediates-related residual antibacterial activities, and (3) to initiate as well as advance our understanding of ENR degradation at genetic level with potential catabolic genes revealed via analysis of comparative transcriptome data.

Chemicals and media

Enrofloxacin (ENR, 98%), ciprofloxacin (CIP, ≥ 98%), norfloxacin (NOR, 98%), and ofloxacin (OFL, 98%) were purchased from Aladdin Industrial Corporation. Malachite green (MG, 98%) and leucomalachite green (LMG, 98%) were purchased from Sinopharm Chemical Reagent Co., Ltd. Chromatographic grade acetonitrile was purchased from Sigma-Aldrich (St. Louis, USA). All the other chemicals and solvents obtained from Sinopharm Chemical Reagent Co. Ltd. (China) were of analytical grade. Molecular biology reagents were purchased from Nanjing Nazyme Biotech Co., Ltd. (China).

Mineral salt medium (MM) contained 1.0 g L^− 1 NH₄NO₃, 1.5 g L^− 1 K₂HPO₄, 0.5 g L^− 1 KH₂PO₄, 1.0 g L^− 1 NaCl and 0.2 g L^− 1 MgSO₄ (pH 7.0 ~ 7.2). Potato-dextrose (PD) media comprised 5.0 g L^− 1 potato extract power and 20.0 g L^− 1 dextrose. Agar (20.0 g L^− 1) was added to the PD medium to make solid media plates (PDA). Oat agar (OA), cornmeal agar (CMA), malt extract agar (MEA), and potato carrot agar (PCA) were used for colony morphology examination and were prepared as described previously (Crous 2009). The lysogeny broth plate (LB) used for the residual antibacterial activity assay contained tryptone (10.0 g/L), yeast extract (5.0 g/L), NaCl (10.0 g/L), and agar (20.0 g L^− 1) at pH 7.0.

Strain isolation and identification

Rhizosphere sediment samples from Vallisneria spiralis L. were collected after our previous phytoremediation experiment (Zhang et al. 2019). Thereafter, 5 grams of the sample were added to a 250 mL Erlenmeyer flask containing 100 mL MM with 1 mg L^− 1 ENR. The flask was then placed on a rotary shaker operating at 160 rpm and incubated in complete darkness at 28°C for a period of 7 days. Following enrichment, 5 mL of supernatant from the culture was added to 100 mL of fresh MM with ENR at equivalent concentration. After three consecutive rounds of enrichment, the enrichment solution was then spread on MM agar plates, containing 1 mg L^− 1 ENR, via gradient dilution. Colonies were picked and purified, before evaluated for their ENR-degrading capability.

Colony morphologies were examined on five types of media: PDA, OA, CMA, MEA, and PCA. Cultures were inoculated in a three-point fashion and incubated in the dark at 25°C for 7 days. Colony diameters were measured, and the colors were described according to mycological color charts (RW 1970). The micromorphological description was mainly based on cultures grown on PDA. The mycelium and spores underwent staining with lactophenol blue and were subsequently observed through a light microscope (OLYMPUS®) at a magnification of 640× (16 × 40). Further identification was carried out via analysis of tandem conserved sequences. To this end, three conserved sequences, ITS&LSU, rpb2, and tub2, serving as representatives of the family Cheatomiaceae, were amplified using the relevant primer pairs of ITS5/NL4, rpb2-5F2/rpb2AM-7R, and T1/TUB4Rd, respectively (Table S1). The PCR conditions used were identical to those described by Wang et al. (2016). Each amplicon was purified and sequenced. Sequence alignment was carried out in comparison with thirty-eight other similar and homologous sequences (Table S2) retrieved from the European Nucleotide Archive (ENA) or GenBank portal using the ClustalX tool in MEGA 5 Software (Larkin et al. 2007, Tamura et al. 2011). Phylogenetic typing analyses were conducted using the neighbor-joining tree method.

Biodegradation experiments

A suspension of conidia from strain KC0924g was prepared and adjusted to a concentration of approximately 4.5 × 10⁶ conidia mL^− 1. The conidial suspension was then inoculated at 5‰ (V/V) into a 150 mL flask containing 50 mL of PD medium and incubated at 28°C and 160 rpm until mycelial pellet formation (approximately 36 h). To assess the influence of Cu²⁺ on ENR degradation by strain KC0924g, a final concentration of 500 µM Cu²⁺ was added to the culture and allowed to incubate for 24 h. Subsequently, the mycelial pellets were collected via centrifugation (6000 rpm, 5 min), washed twice with MM, and then resuspended in 20 mL MM to create resting cells.

For all the biodegradation experiments, unless stated otherwise, the resting cells were inoculated at approximately 2 mg (dry weight) per mL into a 150 mL Erlenmeyer flask that contained 50 mL of MM with 1 mg L^− 1 ENR. The cultures were incubated at 28°C and 160 rpm on a rotary shaker. Specific samples were collected at predetermined times (0 h, 6 h, 12 h, 24 h, 48 h, 72 h, 120 h, and 168 h), and the amount of residual ENR was assessed using high-performance liquid chromatography (HPLC) with fluorescence detection (FLD). To assess the effect of temperature on degradation, cultures were incubated at temperatures of 15, 20, 28, 35, and 40°C. The effect of initial pH on degradation was assessed by adjusting the pH of the medium to 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0. Strain KC0924g was inoculated into MM supplemented with 1 mg L^− 1 CIP, NOR, OFL, MG, and LMG to assess its protential for degrading other FQs, MG and LMG. The control was inoculated with resting cells that had been deactivated by heat. All tests were conducted in triplicate on a rotary shaker (160 rpm) and kept in the dark to prevent photodegradation. Sample cultures were centrifuged and filtered through a 0.22 µm membrane filter to remove impurities and biomass and were then stored at 4°C for further analysis.

The degradation rate (Y) of the compounds mentioned above was quantified using the following equations:

Y (%) = 100 × (C_CK − C_T)/C_CK

where C_CK refers to the concentration of the compound in the control test, and C_T pertains to the concentration of the compound in the treatment test.

A first-order kinetic model was created to elucidate the degradation efficiency of ENR by strain KC0924g with or without Cu²⁺ treatment.

ln (C₀ / C_t) = kt

t_1/2=ln 2/k

where C₀ and C_t refer to the concentration (mg L^− 1) of ENR at time zero and time t, respectively; k represents the ENR degradation rate constant (h^− 1); and t_1/2 (h) denotes the half-life time for ENR degradation.

Chemical analysis

The FQs concentrations present in the cultures were measured through the use of an Agilent 1260 HPLC system equipped with an Eclipse Plus C₁₈ reversed-phase column (250 mm×4.6 mm, 5 µm particle sizes, Agilent) along with fluorescence detection. This method utilized the retention time and peak area of pure standards, and had an excitation wavelength of 280 nm and emission wavelength 450 nm for ENR, CIP, and NOR, and an excitation wavelength 283 nm and an emission wavelength of 490 nm for OFL. The mobile phase consisted of phosphoric acid-triethylamine (0.05 M, pH 2.4) and acetonitrile (18:82, v/v). The flow rate used was 1.0 mL min^− 1.

The metabolites were detected using ultra-performance liquid chromatography coupled with a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific), which was equipped with an electrospray ionization (ESI) probe. The UPLC-HRMS parameters were as follows: the column used was Hypersil GOLD C₁₈ column (100 mm × 2.10 mm, particle size: 3 µm, Thermo Fisher Scientific), the flow rate was set at 0.2 ml min^− 1, and injection volume at 5 µL with mobile phase comprising of solvents A (ultrapure water containing 0.1% v/v formic acid) and B (acetonitrile). Separation was conducted for 30 min under these conditions. 0 ~ 4 minutes: Maitain the volume ratio of solution B at 30%. 4 ~ 21 minutes: Increase the volume of solution B from 30–95%. 21 ~ 25 minutes: Keep the volume of solution B at 95%. 25 ~ 30 minutes: Reduce the volume ratio of solution B back to 30%. The ionization method used was positive ion, and the ion transmission temperature, source voltage, protective gas, and auxiliary gas were 300°C, 4 kV, 35 arb, and 10 arb, respectively. The data analysis software employed was Xcalibur.

Solid-phase extraction combined with high-performance liquid chromatography-tandem mass spectrometry (SPE-HPLC‒MS/MS) was used to detect residues of MG and LMG in the cultures. Following the manufacturer's guidelines, MG and LMG were extracted, enriched, and eluted using PRS columns (Thermo Fisher Scientific). The HPLC‒MS/MS conditions were as follows: Column: Thermo C18 chromatographic column (100 mm×2.1 mm); The separation process was executed at a flow rate of 0.2 mL/min with an injection volume of 10 µL. The mobile phase was composed of acetonitrile solvents B and 0.2% ammonium acetate solvent D. The process was carried out for 10 mins, with the flow rate at 0.2 mL/min. 0 ~ 1.50 minutes: Increase the volume of solution D from 0 to 35%. 1.50 ~ 1.51 minutes: Decrease the volume of solution D from 35–10%. 1.51 ~ 4.50 minutes: Maintain the volume ratio of solution D at 10%. 4.50 ~ 4.51 minutes: Increase the volume of solution D from 10–35%. 4.51 ~ 9.00 minutes: Maintain the volume ratio of solution D at 35%. 9.00 ~ 10.00 minutes: Reduce the volume ratio of solution D to 0%.

Residual antibacterial activity assay

The antibacterial activity of degradation products of FQs against nonresistant Escherichia coli DH5α (Gram-negative) was evaluated using the agar plate diffusion method (punch method). In brief, 500 µL of log-phase E. coli DH5α culture was spread on LB plates. Six dispersed wells were then drilled in each plate using a sterile 8 mm diameter punch. Three of the six wells in each plate were injected with the FQs solution and the other three with the corresponding degradation product solution. Each sample was injected into the well in a volume of 200 µL and the plates were incubated in a constant temperature incubator at 37°C for 20 hours. The inhibition circle diameter (Φ) was measured using a digital vernier caliper. The relative inhibition rate (R) of the degradation product was evaluated by comparing the inhibition zone sizes of the product and drug solutions.

R (%) = 100 × (Φ_p-8)/(Φ_m-8)

where Φ_p and Φ_m represent the inhibition circle diameter of degraded product and FQs drug, respectively.

Comparative transcriptome analysis

Sample preparation, RNA extraction, cDNA library construction, and Illumina HiSeq sequencing

Mycelial cells with or without Cu²⁺ treatment for comparative transcriptome sequencing were obtained as described in 2.3. The obtained cells were immediately washed three times with sterile PBS buffer and stored at -80°C before further treatment. Each treatment was performed in three independent experiments. Total RNA was extracted from mycelia using TRIzol® reagent according to the manufacturer’s instructions (Invitrogen), and genomic DNA was removed using DNase I (TaKaRa). RNA quality was then determined using a 2100 Bioanalyzer (Agilent) and quantified using an ND-2000 (NanoDrop Technologies). High quality RNA samples (OD260/280 = 1.8 ~ 2.2, OD260/230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0, > 10 µg) were used to construct a sequencing library. The cDNA library was prepared using a TruSeqTM RNA Sample Preparation Kit from Illumina (San Diego, CA) and sequenced using an Illumina NovaSeq 6000 sequencer (150 bp*2, Shanghai BIOZERON Co., Ltd.).

De novo assembly, annotation, and differential expression analysis

Raw paired-end reads were trimmed and quality controlled using Trimmomatic with parameters (http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic). RNA de novo assembly was performed using Trinity (http://trinityrnaseq.sourceforge.net/) with clean data from from all samples. All the assembled transcripts were searched against the NCBI Protein Nonredundant (NR), String, and KEGG databases using BLASTX to identify proteins with the highest sequence similarity to the given transcripts and to retrieve their functional annotations. The expression level of each transcript was calculated using the reads per kilobase of exon per million mapped reads (RPKM) method, and differentially expressed genes (DEGs) with a P value < 0.05 and |log2 (fold change)| (|log2FC|) ≥ 2 were selected by comparing the CU and CK groups. Finally, the DEGs were annotated and analyzed in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases.

Data analysis and nucleotide sequence accession numbers

All experiments were repeated three times, and the data are presented as the mean ± standard deviation. Differences between treatments were analyzed by one-way ANOVA and Duncan’s multiple range tests at P < 0.05 using IBM SPSS Statistics 26 (IBM Crop., Armonk, NY, USA).

The GenBank accession numbers for the ITS, ITS&LSU, rpb2, and tub2 gene sequences of strain KC0924g were ON055749, ON025176, ON033740, and ON033741, respectively. The raw sequence reads for the transcriptome of strain KC0924g have been submitted to the NCBI Sequence Reads Archive (SRA) database under the SRA accession number: PRJNA1022865.

A filamentous fungal strain, KC0924g, exhibiting a high ENR degradation ability, was isolated from the rhizosphere sediment sample. Colonies of strain KC0924g on PDA were 47 to 58 mm in diameter after 7 days at 25°C, with entire edges. The aerial mycelium was thick, white to yellow and olivaceous black in reverse (Fig. 1a). The colony morphologies of strain KC0924g grown on four other different media (OA, CMA, MEA, and PCA) are shown in Fig. S1, and their characteristics are described in the figure legend. Strain KC0924g possessed thick-walled conidia and an acremonium-like synanamorph (Fig. 1bc). The above morphological characteristics of strain KC0924g were similar to those of the model strain Humicola pulvericola CBS 723.97^T (Wang et al. 2019). A BLASTN search against the sequences on the National Center for Biotechnology Information (NCBI) website revealed that the ITS gene sequence of strain KC0924g shared 98.99% similarity to Humicola sp. JS-0112 and Chaetomium sp. TR160. Hence, the rDNA-ITS sequence alone cannot distinguish the taxonomic status of strain KC0924g. It was found that the 5.8S nrRNA gene region and the D1/D2 domains of the 28S nrDNA (ITS&LSU), the second largest subunit of the DNA-directed RNA polymerase II gene region (rpb2) and the partial beta-tubulin gene region (tub2) were used as DNA barcodes to differentiate Humicola species and related genera in Chaetomiaceae (Wang et al. 2016). Subsequently, the phylogenetic tree (Fig. 1d) built based on the ITS&LSU-rpb2-tub2 gene sequences of strain KC0924g and related strains revealed that strain KC0924g clustered with Humicola pulvericola CBS 723.97^T (Wang et al. 2019). Combining the morphological characteristics with phylogenetic analysis, the ENR degrader was identified as a member of the genus Humicola and deposited at the China Center for Type Culture Collection (deposition number: CCTCC M 2020752).

Fungi of the genus Humicola are common and widespread and are known to have biotechnological and industrial potential (Ibrahim et al. 2021). In recent years, the genus Humicola has been found to be valuable for environmental remediation. For example, Humicola sp. 2 WS1 showed marked arsenic biomethylation potential. Through bioaugmentation, Humicola sp. 2 WS1 not only attenuated the stressful effects of arsenic on Bacopa monnieri L. in the rhizosphere environment but also promoted the growth of B. monnieri (Tripathi et al. 2020). In this study, Humicola sp. KC0924g was isolated from the rhizosphere sediment sample of the submerged macrophyte V. spiralis. Submerged macrophytes play an essential role in maintaining aquatic ecosystem stability and are widely used in the remediation of contaminated water and sediment (Dai et al. 2014). Further work is required to explore the effect of Humicola sp. KC0924g and V. spiralis interaction on sediment remediation. Admittedly, this is the first report that a Humicola species can degrade the antibiotic ENR.

Degradation of ENR by strain KC0924g

Temperature and pH are two critical environmental factors that impact xenobiotic degradation. This is due to the influence of temperature on the growth of microorganisms and the activity of certain catabolic enzymes, as well as the impact of the initial pH of the medium on the movement of compounds to microorganisms and the alteration in the solubility of contaminants. Therefore, the effects of different temperatures and the initial pH value of the medium on ENR degradation were studied separately. Among five temperatures ranging from 15°C to 40°C, the optimum degradation temperature was observed at 28°C with maximum degradation of 89.6%. When the temperature lowered to 15°C, the rate of degradation decreased to 69.4%. Subsequently, with an increase in the temperature to 40℃, the degradation efficiency surpassed 76.9%. Overall, Humicola sp. KC0924g exhibited relatively broad temperature adaptability for ENR degradation. The degradation rate of ENR affected by temperature was listed in the following order: 28℃>20℃≥35℃>40℃>15℃ (Fig. 2a). The optimum temperature for photosynthesis in temperate submerged plants is usually between 25 ~ 32°C (Barko et al. 1982, Santamaría and van Vierssen 1997, Pedersen et al. 2013). As can be seen from the results, the optimal temperature interval for ENR degradation by Humicola sp. KC0924g was consistent with that for the growth of temperate submerged plants. Subsequently, the effect of different pH values (ranging from 5.0 to 11.0) on the degradation of ENR by Humicola sp. KC0924g was investigated at the optimum temperature of 28°C. The degradation rate was not significantly different when the initial pH was 7.0, 8.0 and 9.0 (p>0.05), and the maximum degradation rate was as high as 98.2% at pH of 9.0. The degradation rate decreased significantly when the initial pH was below 6.0 or above 10.0. The degradation extents of ENR affected by pH were ranked in the following order: pH9.0 ≥ pH8.0 ≥ pH 7.0>pH 10.0>pH 6.0 (Fig. 2b). The above experiments showed that this strain could degrade ENR efficiently under neutral to slightly alkaline conditions. The optimal pH range required for fish and shrimp growth in aquaculture is between 7.5 and 8.0 (Liu and Wang 2012). During this range, ENR can be efficiently degraded by strain KC0924g. The dynamic degradation curve of ENR (initial concentration of 1 mg L^− 1) under the optimal condition (28°C and pH 7.0) by strain KC0924g is shown in Fig. 2c. The degradation rate increased immediately during the first 12 h, then gradually slowed down between 12 h and 72 h and remained stable after 72 hours.

Identification of the metabolites and proposed ENR degradation pathways

Nine metabolites were preliminarily detected in the catabolic supernatant by means of ultra-performance liquid chromatography coupled with high-resolution electrospray ionization mass spectrometry (UPLC-HRMS) (shown in Fig. 3). All identified peaks were found to correspond to protonated derivatives of the theoretical values of the corresponding compounds with errors between − 1.39 and 0.04 ppm (shown in Table 1). In contrast, no corresponding mass spectrum peaks of these metabolites were found in the control. Four metabolic pathways were proposed based on the identified metabolites (Fig. 4). In Pathway I, ENR underwent transformation through the splitting of the piperazine moiety, resulting in the production of Compounds C1 and C2. According to Adjei et al. (2006), it was believed that the antibacterial activities of Compounds C1 and C2 were reduced due to the cracking of the piperazine ring. In Pathways II, III, and IV, ENR underwent biotransformation through hydroxylation of the aromatic core, oxidative decarboxylation, and oxidative defluorination to generate C3, C4, and C5, respectively. For C5, further pathways existed. These included pathway iv1 and pathway iv2. In pathway iv1, C6 was created by further hydroxylation of C5 at the aromatic core. In pathway iv2, C7 was produced by oxidative removal of the cyclopropyl group from the quinoline ring of C6. Afterward, the piperazine ring of C7 slowly degraded through C-N bond cleavage, yielding C8 and C9.

Table 1

Spectral data for ENR and postulated biodegradation metabolites determined from UPLC-HRMS
Compound	Peak mass [M + H]⁺(m/z)	Display Formula	Theo. mass [M + H]⁺(m/z)	Delta (ppm)*	RT (min)	References
ENR	360.1713	C₁₉H₂₃O₃N₃F	360.17180	-1.39	11.25
C1	334.1559	C₁₇H₂₁O₃N₃F	334.15615	-0.88	3.99	(Wetzstein et al. 1997, Karl et al. 2006)
C2	263.0826	C₁₃H₁₂O₃N₂F	263.08265	-0.14	1.81
C3	376.1663	C₁₉H₂₃O₄N₃F	376.16671	-1.20	1.21
C4	332.1768	C₁₈H₂₃O₂N₃F	332.17688	-0.19	1.11
C5	358.1761	C₁₉H₂₄O₄N₃	358.17613	0.04	3.69
C6	374.1708	C₁₉H₂₄O₅N₃	374.17105	-0.64	0.83
C7	318.1446	C₁₆H₂₀O₄N₃	318.14483	-0.66	3.10	This study
C8	249.0870	C₁₂H₁₃O₄N₂	249.08698	-0.09	12.41	This study
C9	235.0713	C₁₁H₁₁O₄N₂	235.07133	-0.25	10.91	This study

*Generally, a mass error between − 5 ppm and 5 ppm is acceptable (Blake et al. 2011).

Previous studies have shown that basidiomycetes fungi decay wood and degrade certain xenobiotics due to their ability to produce hydroxyl radicals (H₂O₂), which trigger an extracellular Fenton-type reaction(Jensen et al. 2001). A total of 137 ENR metabolites produced by Gloeophyllum striatum and other basidiomycetous fungi were identified, in which the principal routes for ENR metabolism include hydroxylation of the aromatic core, oxidative decarboxylation or defluorination, or decomposition of the piperazine moiety and N-oxidation (Wetzstein et al. 1997, Karl et al. 2006, Wetzstein et al. 2006, Parshikov and Sutherland 2012). In this study, ENR major metabolites catalyzed by the ascomycete fungi Humicola sp. KC0924g were similar but differed slightly from those of Gloeophyllum striatum. The presence of the fluorine atom at the R6 position, the piperazine ring at the R7 position, and the cyclopropyl group at the R1 position in FQs improves the antibacterial effect of the drug and reduces the compatibility of the drug with microbial degradation (Van Caekenberghe and Pattyn 1984, Bhatt and Chatterjee 2022). In Pathway IV to iv2, ENR first underwent hydroxyl substitution to remove the fluorine group, then the cyclopropyl group was removed, and finally, the piperazine ring gradually cracks. As the three essential functional groups in the ENR molecule were consecutively degraded, we speculated that this pathway might diminish the antibacterial efficacy of ENR while enhancing its degradability. However, due to the low concentration of the metabolites, the information obtained from UPLC-HRMS alone is not sufficient to fully analyze their chemical structures. The exact stereochemistry of these trace metabolites will be determined in future work by enriching and purifying the metabolites followed by NMR spectroscopy.

Degradation of other fluoroquinolones, malachite green, and leucomalachite green by strain KC0924g

Strain KC0924g showed degradative capabilities against additional types of FQs, such as including CIP, NOR, and OFL. The removal rates of CIP, NOR, and OFL by strain KC0924g after 72 h of incubation were 86.4%, 70.2%, and 46.4%, respectively (Fig. 2d). It was found that the different types of FQs have similar molecular configurations and follow similar biodegradation pathways, but slight differences in their molecular structures or different toxicities to microorganisms could lead to altered degradation rates (Parshikov and Sutherland 2012, Bhatt and Chatterjee 2022). FQs have similar chemical structures but different toxicities to microorganisms, which may explain why KC0924g is able to degrade different classes of FQs at different rates.

Furthermore, strain KC0924g could also degrade MG and LMG (Fig. 2d and Fig. S2). The degradation rate of MG reached 99.9% without accumulating LMG and that of LMG reached 87.5%. MG is a synthetic triphenylmethane dye with strong bactericidal, fungicidal and parasiticidal properties (Srivastava et al. 2004). As a result, MG was once extensively applied in fisheries to prevent and control diseases, such as saprolegniasis, white spot disease, and ciliate disease (Bergwerff and Scherpenisse 2003, Srivastava et al. 2004). LMG is the major reductive metabolite of MG, which persists in fish tissues and the environment for long periods of time (Li et al. 2012). The use of MG in aquaculture is now strictly prohibited as MG and LMG have been shown to be toxic and carcinogenic (Lin et al. 2016). However, residues of MG and LMG are often detected in the aquaculture environment. Therefore, MG must be removed from the environment to prevent potential harm to human health. The ability of strain KC0924g to degrade many common contaminants in the aquaculture environment makes it a good candidate for environmental remediation.

Residual antibacterial activity assay

The antibacterial activity of the supernatants against the gram-negative bacterium E. coli DH5α (non-resistant) was measured directly by means of the agar diffusion method. The residual antibacterial activity was expressed as a percentage of the initial drug (1 mg L^− 1). After 72 hours of degradation by strain KC0924g, the antibacterial activities of the four FQs (ENR, CIP, NOR and OFL) in the supernatants decreased by approximately half, with residual antibacterial activities of 51.57%, 56.63%, 37.73% and 44.72%, respectively (shown in Fig. 5). In addition, a control with the supernatant of strain KC0924g after 72 h incubation in MM medium (without FQs) showed no antibacterial activity against E. coli DH5α, ruling out the effect of endogenous fungal antimicrobials in this experiment. Parent drugs that have not been completely degraded and degradation products with antimicrobial groups (e.g. piperazine groups) may account for the residual antibacterial activity in the supernatants. Čvančarová et al. (2015) showed that the supernatant of FQs degraded by ligninolytic fungi still exhibited high residual antibacterial activity against gram-negative and gram-positive strains that were not resistant to FQs in the environment. Wetzstein et al. found that the residual antibacterial activity of CIP supernatant against gram-negative Escherichia coli ATCC 8739 was attenuated after 13 weeks of degradation by species of basidiomycetes (Wetzstein et al. 1999). After degradation by a Thermous sp. isolate, the residual antibacterial activity of the supernatant of four FQs against Escherichia coli K12 decreased by 20–40% (Pan et al. 2018).

Cu²⁺ treatment increased the efficiency of ENR removal by strain KC0924g

It was observed that resting cells of strain KC0924g treated with Cu²⁺ displayed a higher efficiency of ENR removal in comparison to untreated resting cells. The removal of ENR by cells of strain KC0924g with or without Cu²⁺ treatment could be fitted by the first-order kinetic model. Table 2 presents the values for k (0.0308, 0.0763) and R² (0.931, 0.969), demonstrating that the first-order kinetic model corresponds to the data well. The half-life of ENR (1 mg L^− 1) measured in Cu²⁺-treated cells reached 3.9 h, compared to 9.8 h obtained in untreated cells. Moreover, the removal rate of ENR in Cu²⁺-treated cells within 24 h reached 98.9%, a higher value than the 77.39% measured in untreated cells. The negligible change in ENR concentration upon treatment with Cu²⁺ alone (in the absence of microorganisms) confirmed that microorganisms dominate ENR degradation. The results showed that the addition of Cu²⁺ could greatly improve the degradation efficiency of ENR by strain KC0924g. This study is consistent with some previous studies indicating that low levels of Cu²⁺ may promote xenobiotic degradation. For example, Xu and Wang (2014) found that low concentrations of Cu²⁺ stimulated the degradation efficiency of decabromodiphenyl ether by the white rot fungus Phlebia lindtneri JN45(Xu and Wang 2014). Similarly, Sangare et al. (2014) demonstrated that Cu²⁺ is an activator of aflatoxin B1 degradation by Pseudomonas aeruginosa N17-1(Sangare et al. 2014).

Table 2

Kinetic parameters and removal of ENR (1 mg L^− 1) by strain KC0924g (induced by Cu²⁺) after a 24 h incubation
Cu²⁺ (µM)	k (h^− 1)	T_1/2 (h)	R²	Removal (%)
0	0.0308	9.8	0.931	77.39
500	0.0763	3.9	0.969	98.94
k-Kinetic removal rate constant (h^− 1);
T_1/2-Degradation half-life (h);

R²-correlation coefficient.

Comparative transcriptome analysis of strain KC0924g with and without Cu²⁺ treatment

Six libraries were constructed from mRNA of control (CK) and Cu²⁺-treated (CU) samples to investigate the transcriptomic responses to Cu²⁺ in strain KC0924g. These libraries each generated approximately 53 million, 74 million, 53 million, 53 million, 57 million and 51 million raw reads, and after quality control, over 88% and 83% of clean reads were obtained from CK samples (CK-1, CK-2 and CK-3) and Cu²⁺-treated samples (CU-1, CU-2 and CU-3), respectively (Table S3). After de novo assembly using Trinity software, 24531 unigenes were obtained, with an average length of 1513 bp and an N50 of 2519 bp. The number and percentage of unigenes with significant similarity to sequences in the NR, GO, COG, KEGG and SWISS-prot databases are listed in Table S4. Generally, 21,527 unigenes were annotated in at least one database, accounting for 87.75%. Comparative transcriptome analysis was performed to explore the differentially expressed genes (DEGs) between the CK and CU groups. A total of 11570 genes were identified as DEGs in one group relative to the other group. In the CU group, 275 genes were upregulated and 11,295 genes were downregulated compared to the CK group (Fig. 6a, b), indicating that Cu²⁺ has a repressive impact on the expression of the majority of DEGs. Gene Ontology (GO) enrichment analysis was conducted to identify the primary functional categories of DEGs. As shown in Fig. 6c and Table S5, the DEGs were mainly enriched in the GO categories of biological processes (BP) and cellular component (CC). The top three enriched items were plastid (genes number = 1509, cellular component category, GO:0009536), chloroplast (genes number = 1481, cellular component category, GO:0009507) and developmental process (genes number = 1361, biological processes category, GO:0032502). The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the DEGs identified 37 significantly enriched pathways (P<0.05), of which ribosome (ko03010), carbon metabolism (ko01200) and protein processing in the endoplasmic reticulum (ko04141) were the top three enriched pathways (Fig. 6d and Table S6).

Most of the detected DEGs involved in xenobiotic biodegradation and metabolism were downregulated. However, only two genes encoding FAD-dependent monooxygenase (TRINITY_DN2180_c0_g1:K00480) and a hypothetical protein (TRINITY_DN13314_c0_g1) were found to be upregulated (Table S7). FAD-dependent monooxygenase (FMO), an oxidoreductase with flavin adenine dinucleotide or flavin mononucleotide as a coenzyme, is involved in diverse biochemical redox reactions and the synthesis of intricate natural products (van Berkel et al. 2006, Huijbers et al. 2014). In nature processes, FMO catalytic activity initiates the breakdown of xenobiotics, and bacteria have also used this catalytic activity to degrade or inactivate clinically used antibiotics(Reis et al. 2021). A class A FMO, bacterial tetracycline destructase (TetX), has been discovered to hydroxylate the C11a of tigecycline, causing it to become inactivated by producing 11a-hydroxytigecycline (Volkers et al. 2011). Rifampicin monooxygenase (Rox) is another class A FMO that was propsed to catalyze the hydroxylation of C2 in rifampicin, resulting in the generation of 2’-N-hydroxy-4-oxo-rifampicin based on an NMR analysis of the Rox crystal structure(Hoshino et al. 2010). On the other hand, BVMO (Baeyer-Villiger monooxygenase) is a class B FMO that has been discovered to oxidize the carbonyl moiety of the β-lactam ring (Minerdi et al. 2016). Molecular docking studies have identified active sites within the crystal structure that interact with vancomycin and/or methicillin (Hwang et al. 2018). Furthermore, the sulfonamide monooxygenase (SadA/SadC) constitutes a two-component FMO system that facilitates the conversion of sulfamethoxazole to products that lack antimicrobial activity (Ricken and Kolvenbach 2017). The upregulated FMO (TRINITY_DN2180_c0_g1) in this research probably accommodates the ENR for oxidative reactions. Nonetheless, further investigation is necessary to unravel the precise role of this FMO.

A previous report indicated that laccase and cytochrome P450 enzymes play roles in FQs degradation by white-rot fungi (Prieto et al. 2011, Gao et al. 2018). Through transcriptome analysis, we found that only one laccase gene (TRINITY_DN6638_c0_g1) was upregulated in the CU group, and no significantly upregulated genes encoding cytochrome P450 enzymes were found in the same group. The BLAST results indicated that the amino acid sequence similarity of this particular sequence with the laccase sequences from Trametes versicolor and Phanerochaete chrysosporium were 24.26% and 25.99%, respectively (Fig. 7). For comparison, the amino acid sequence similarity of the laccases from Trametes versicolor and Phanerochaete chrysosporium was 26.91%, and both laccases have been previously observed to biotransform FQs. The laccase activity of strain KC0924g was not detected, possibly due to low quantity and activity of extracellular laccase. Futher attempts to increase the laccase concentration will be made to study its catalytic function. The transcriptome analysis of strain KC0924g highlighted potential biodegradation-related enzymes, which could assist in future investigation of the microbial degradation molecular mechanism of FQs.

Humicola sp. KC0924g was isolated for its ability to degrade ENR from rhizosphere sediments from a previous phytoremediation experiment. The optimum conditions for ENR degradation by strain KC0924g were at 28°C and pH of 9.0. The biodegradation metabolites were detected and identified using UPLC-HRMS, and four distinct pathways were proposed. Additionally, strain KC0924g demonstrated the ability to decompose MG, LMG and three other types of FQs. The degradation efficiency of ENR by strain KC0924g can be significantly improved by Cu²⁺. An FMO and a laccase were predicted to be involved in the degradation process of ENR based on comparative transcriptomic analyses. The successful isolation of strain KC0924g offers a potential candidate strain for bioremediation applications and an opportunity for further investigation into the interactions between fungi and plants in the rhizosphere, as well as subsequent clues to the molecular mechanisms of ENR degradation.

Ethics approval and consent to participate: Not applicable.

Consent for publication: All authors have read the manuscript and agreed to its publication.

Availability of data and material: Not applicable.

Competing interests: The authors declare no conﬂicts of interest.

Funding: This research was supported by the Shanghai Agriculture Applied Technology Development Program, China (Grant No. 2020-02-08-00-07-F01483), the National Natural Science Foundation of China (31900097), and the Natural Science Foundation of Shanghai (20ZR1450800).

Authors' contributions: Xueting Chen and Yuping Zhang conceived and designed the experiments; Xueting Chen and Jinghua Liu performed the experiments and analyzed the data; Xueting Chen wrote and submitted the paper. All authors have read and agreed to the published version of the manuscript.

Acknowledgements: This research was supported by the Shanghai Agriculture Applied Technology Development Program, China (Grant No. 2020-02-08-00-07-F01483), the National Natural Science Foundation of China (31900097), and the Natural Science Foundation of Shanghai (20ZR1450800).

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A novel enrofloxacin-degrading fungus, Humicola sp. KC0924g, isolated from the rhizosphere sediment of the submerged macrophyte Vallisneria spiralis L.

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Journal Publication

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Abstract

Figures

Introduction

Materials and Methods

Chemicals and media

Strain isolation and identification

Biodegradation experiments

Chemical analysis

Residual antibacterial activity assay

Comparative transcriptome analysis

Sample preparation, RNA extraction, cDNA library construction, and Illumina HiSeq sequencing

De novo assembly, annotation, and differential expression analysis

Data analysis and nucleotide sequence accession numbers

Results and DiscussionIsolation and identification of the ENR-degrading fungus

Degradation of ENR by strain KC0924g

Identification of the metabolites and proposed ENR degradation pathways

Degradation of other fluoroquinolones, malachite green, and leucomalachite green by strain KC0924g

Residual antibacterial activity assay

Cu²⁺ treatment increased the efficiency of ENR removal by strain KC0924g

Comparative transcriptome analysis of strain KC0924g with and without Cu²⁺ treatment

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

A novel enrofloxacin-degrading fungus, Humicola sp. KC0924g, isolated from the rhizosphere sediment of the submerged macrophyte Vallisneria spiralis L.

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and Methods

Chemicals and media

Strain isolation and identification

Biodegradation experiments

Chemical analysis

Residual antibacterial activity assay

Comparative transcriptome analysis

Sample preparation, RNA extraction, cDNA library construction, and Illumina HiSeq sequencing

De novo assembly, annotation, and differential expression analysis

Data analysis and nucleotide sequence accession numbers

Results and DiscussionIsolation and identification of the ENR-degrading fungus

Degradation of ENR by strain KC0924g

Identification of the metabolites and proposed ENR degradation pathways

Degradation of other fluoroquinolones, malachite green, and leucomalachite green by strain KC0924g

Residual antibacterial activity assay

Cu2+ treatment increased the efficiency of ENR removal by strain KC0924g

Comparative transcriptome analysis of strain KC0924g with and without Cu2+ treatment

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Cu²⁺ treatment increased the efficiency of ENR removal by strain KC0924g

Comparative transcriptome analysis of strain KC0924g with and without Cu²⁺ treatment