Effects of colistin stress on growth, antioxidant system and metabolite pattern of lettuce

Abstract

Background

The widespread of colistin resistance gens has aroused great public concern, and many efforts have been made to re-evaluate the application of colistin in veterinary medicines. Most of colistin is excreted with feces, while no literature has reported the determination of colistin in plants and its accumulation on the potential toxicity to plant life. In this study, a rapid and eco-friendly method using liquid chromatography tandem mass spectrometry (LC-MS/MS) based on dispersive solid-phase extraction was developed for detecting colistin in lettuce and was successfully applied to real samples. Furthermore, the toxic effects of colistin exposure on the growth, oxidative stress, and metabolism of lettuce were investigated.

Results

The LC-MS/MS results showed that the developed method had good linear correlation. Average recoveries of colistin at four spiked concentrations (10, 30, 100, and 300 µg/kg) ranged from 73.3% to 91.1%, and the limit of detection (LOD) was 5 µg/kg. Colistin may stimulate lettuce seed germination, although the difference was not significant; however, it can significantly inhibit lettuce growth in a dose-dependent manner. Colistin can damage mitochondria and chloroplasts, blocking photosynthetic efficiency and energy metabolism, thereby inhibiting lettuce growth. This harmful effect is probably associated with the suppression of the phospholipid-arachidonic acid metabolic pathway, during which colistin upregulated 11 (R)-HETE and reduced 5,6-DHET and prostaglandin G2. Consequently, malondialdehyde, peroxidase, and superoxide dismutase were significantly elevated, while catalase was diminished, triggering lipid peroxidation and the excessive accumulation of oxidative stress indicators. Finally, based on detection results from real samples collected at local supermarkets, the hazard quotient was below 1, indicating a low health risk of colistin in lettuce for local residents through dietary intake.

Conclusion

This study developed a rapid LC-MS/MS method for the detection of colistin in lettuce. Colistin was found to upregulate 11 (R)-HETE and decrease 5,6-DHET and prostaglandin G2 by inhibiting the phospholipid-arachidonic acid metabolic pathway, thereby inducing oxidative stress and compromising organelle integrity in lettuce. Analysis of real samples indicated that the potential health risk from colistin exposure through dietary intake is low. Nevertheless, further research is required to evaluate the potential risks related to antibiotic resistance and the synergistic toxicity of colistin in combination with other contaminants.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-025-07885-w.

Introduction

Colistin is a polypeptide antibacterial agent produced by Bacillus polymyxins, which mainly damages the cell wall of gram-negative bacteria through increasing membrane permeability [1]. Colistin has been used to treat gram-negative bacterial infections for a long time, especially to treat serious infections caused by multidrug-resistant (MDR) microorganisms such as Acinetobacter baumannii and Pseudomonas aeruginosa, so called “the last-defense antibiotic” [2]. However, the extensive use of colistin in livestock exacerbates the selection and transmission of drug resistance between animals and humans. In particular, the colistin resistance gene (MCR-1) has been spread in humans and animals around the world since it was first identified in China in 2015 [3]. It is urgent to control or regulate the use of colistin in animal production to limit the development of drug-resistant bacteria. Therefore, the addition of colistin to feed for animal growth promotion has been banned since 2016 in China. Colistin cannot be absorbed by animal’s body, and it is excreted as the prototype drug through animal wastes (urine and feces). Colistin can contaminate soil, surface water, and groundwater following the release of untreated agricultural waste. It has been detected in animal feces and surrounding soil at concentrations up to 17.0 mg/kg, potentially leading to drug residues in vegetables [4]. In addition to exacerbating drug resistance, colistin accumulated in vegetables has toxic effects such as ototoxicity and nephrotoxicity, posing a threat to human health through the food chain. At present, many studies have been reported on the determination of colistin residue in animal-derived products or feeds [5], but no report on the analysis of vegetable samples. Accordingly, it is necessary to construct a method for the determination of colistin in vegetables.

The extensive use of antibiotics as feed additives for animal production has increased residual risk in the environment. Numerous studies have confirmed that plants can absorb antibiotics, leading to harmful effects on their growth [6, 7]. The toxic effects are multifaceted, including inhibiting seed germination, slowing rhizome growth, and reducing chlorophyll content. For example, amoxicillin and ampicillin can postpone the germination of rice seed while significantly reducing the germination rate [8]. Although ciprofloxacin did not disrupt germinability of maize seeds, it can significantly decrease the average germination time through promoting reactive oxygen species (ROS) accumulation [9]. Low concentration of sulfamethoxazole (SMZ) can stimulate the growth of C. vulgaris, while 80 ~ 200 mg/L of SMZ exhibited significant inhibitory impact [10]. The mechanisms of toxic effects on plants include cell permeability, antioxidant enzyme activity, and protein expression [2, 11]. Lv et al. found that superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) rose at the beginning and then declined with the increasing concentration of ofloxacin in ginger, probably because of the gradual scavenging of reactive oxygen radicals [12]. Levofloxacin exhibited toxic effects on lupin seedlings through modifying the production of free radicals and protein profile [13]. Another study reported that tetracycline can increase membrane permeability and trigger mitochondrial membrane potential loss of ryegrass [14].

In addition to the commonly used phytotoxicity indices mentioned above, metabolites can directly reflect how plants respond to changes in the environment under drug exposure. As an emerging omics technology, metabolomics has been used to reveal the mechanisms of biochemical processes by analyzing alterations in metabolic products. Currently, certain metabolites are considered vital indicators for assessing comprehensive biological responses. For example, under tetracycline exposure, 45 metabolites were positively correlated with root length of ryegrass through affecting aminoacyl-tRNA biosynthesis, nitrogen metabolism, and alanine, aspartate, and glutamate metabolism [11]. It has been found that seven metabolic pathways were altered in microalgal cells under ciprofloxacin stress, up-regulation of carbohydrate and arachidonic acid metabolism and transport, and enhanced energy production via EMP metabolism as a means of resisting exogenous ciprofloxacin [15].Through nontargeted metabolomics analysis, it was found that rice under the stress of sulfonamide drugs can cause glucose metabolism disorders, mainly starch and sucrose metabolism, leading to the accumulation of small molecule sugars [16]. Numerous studies have reported that antibiotics can alter plant metabolites; however, the potential toxic effects of colistin on plants have not yet been investigated.

In view of the resistance and toxicity problems caused by the extensive use of colistin in livestock farming, research has primarily focused on assessing colistin residues. Considerable efforts have been made to analyze colistin and its resistance gene residues in animal-derived foods and the environment. Lettuce, a commonly consumed vegetable, tends to accumulate higher levels of antimicrobials compared to other crops [17]. However, there are currently no reports on the detection of colistin residues or its toxic effects in lettuce. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of colistin in lettuce to evaluate the potential residual risk to humans. Furthermore, potential effects of colistin on the growth and development of lettuce were evaluated, and the biomolecular mechanism of phytotoxicity was revealed using metabolomics analysis. Finally, based on the real samples, the hazard quotient (HQ) was calculated to evaluate the potential health risk of colistin in lettuce through dietary exposure.

Materials and methods

Reagents and chemicals

The HPLC-grade solvents of formic acid (FA), methanol (MeOH), ammonium hydroxide, ethanol, and acetonitrile (ACN) were purchased from Fisher Scientific (Fairlawn, NJ, USA). Primary secondary amine (PSA) was bought from Welch Materials (Shanghai, China). Colistin was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China), with purity higher than 90%. Microporous membrane filters (0.22 μm) were bought from Jinteng Materials (Tianjin, China).

Standard solutions

To prepare the standard stock solution of colistin (1000 µg/mL), 10 mg of colistin was weighed into a volumetric flask and diluted with water to 10 mL. Standard stock solutions can remain stable for 6 months if they are kept at −20 °C and protected from light. The stock solution was diluted with water to the proper concentration every day to prepare a working standard solution.

The cultivation of lettuce

Fast-growing lettuce (Lactuca sativa var. ramosa Hort.) seeds were purchased from Jiuqiseed Technology Co. (Feidong, Anhui). The seed production and marketing license is D (Anhui Hefei) Nongzhong Xuzi (2020) No. 0001. Table S1 displays the seed quality inspection results, which align with the national standard GB 16715.5–2010. The optimal germination temperature is 15 ~ 20 °C, and the optimal seedling growth temperature is 13 ~ 22 °C. Before the cultivation, lettuce seeds were surface-sterilized by soaking them in 1% sodium hypochlorite for 10 min and then washed with deionized water. The seed germination was investigated according to the report [18]. Briefly, petri dishes (10 cm in diameter) containing one layer of filter paper were used as germination beds, and then 3 mL of different concentrations of colistin solution (10, 30, and 300 µg/mL) were added. The control group received the same volume of deionized water. Each treatment has three biological replicates. Lettuce seeds were cultivated under the following conditions: 20℃ and 80% humidity, 16 h light (illuminance: 6000 lx)/8 h dark-light cycle. Based on the radicle higher than 2 mm, germination energy (GE) was measured on day 3, and germination rate (GR) was determined on day 7.

To accurately reflect the impact of colistin entering the soil via feces on lettuce growth, drug-soil model was constructed. Soil samples were collected from the experimental field at Guizhou University, and were sieved using a 2 mm stainless steel sieve to remove coarse particles (e.g., roots, stones, and leaves). 300 g of soil (containing 150 g of Changbai Mountain organic nutrient soil) was used to cultivate lettuce and the basic properties of soil are as follows: pH = 6.57; carbon = 57.67 g/kg; nitrogen = 3.70 g/kg; phosphorus = 0.40 g/kg; organic matter = 99.43 g/kg, and soil texture class = clay soil. Then, three different concentrations of colistin (10, 30, and 300 µg/kg) were mixed with the soil and deionized water was used instead of colistin in the control group. The bottom of plastic flowerpots (10 × 10 × 8.5 cm) was covered with gauzes on which soil was placed and seeds were placed 2–3 cm deep on the soil surface. Each flowerpot was planted 5 seeds and lettuce seeds were cultivated for 15 days under the following conditions: 20℃ and 80% humidity, 16 h light (illuminance: 6000 lx)/8 h dark-light cycle. Finally, lettuce samples were collected, washed with deionized water, dried with filter paper and stored at −80℃. The content of colistin in lettuce was determined using LC-MS/MS, and microstructure observation, chlorophyll determination, oxidative stress index determination, and metabolomics analysis were performed simultaneously.

Sample Preparation of colistin in lettuce

10 g of homogeneous lettuce was accurately weighed into 50 mL polypropylene centrifuge tubes. Samples were spiked at an appropriate standard solution for incubating 30 min. Colistin was ultrasonically extracted with 10 mL of MeOH-1% FA aqueous solution (1:5) for 10 min and then centrifuged at 8000 rpm for 5 min. A dispersive solid-phase extraction (DSPE) protocol was used for sample purification: 1 mL supernatant was mixed with 1 mL water, and then the mixture was thoroughly blended with 50 mg PSA adsorbent for 2 min. After centrifugation, the extract was filtered through a 0.22 μm filter membrane for LC-MS/MS analysis.

LC-MS/MS analysis of colistin in lettuce

LC-MS/MS analysis was achieved according to the literature. Briefly, a Shimadzu HPLC system (Shimadzu, Kyoto, Japan), which assembled an Applied Biosystems Sciex Triple Quad 5500 triple-quadrupole mass spectrometer, was used. A Phenomenex Luna C₁₈ column (150.0 mm × 2.0 mm i.d., 5.0 μm) was applied to separate colistin. The mobile phase consisted of 0.1% formic acid acetonitrile solution (A) and ACN (B). The gradient elution procedure was as follows: 0.5 min, 95% A; 2.0 min, 70% A; 3.0 min, 20% A; 3.6 min, 20% A; 4.0 min, 95% A; 7.0 min, 95% A. The injection volume was 10 µL, and the flow rate was 0.2 mL/min. The mass conditions were the same as the literature. The MS parameters of colistin were listed in Table S2.

Toxicity assessment of colistin on lettuce growth, pigment and antioxidant system

The chlorophyll concentration was analyzed based on the reported procedures [19]. 0.2 g of fresh leaves of lettuce were picked and cut. Then, the mixture was blended with 10 mL of extract solution (acetone: ethanol = 1:1) and stored in the dark for 24 h. The absorbance value was determined at 663 nm and 645 nm.

To examine ultrastructural alterations in chloroplasts and mitochondria under colistin stress, transmission electron microscopy (TEM) was performed using leaves of 15-day-old lettuce seedlings. Lettuce was fixed with 1% osmium tetroxide for 1 h and then dehydrated stepwise with acetone, followed by infiltration, embedding, and making ultrathin sections, which were subsequently stained with uranyl acetate and lead citrate, and finally, lettuce was placed on a copper grid and observed using a JEM-1400FLASH transmission electron microscope.

Lettuce leaf (1.0 g) was ground in 10 mL cold phosphate buffer (0.1 mol/L, pH 7.0 ~ 7.4). Then, the mixture was centrifuged for 10 min at 5000 r/min (4 °C) and the supernatant was collected for the determination of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and malondialdehyde (MDA). The SOD, POD, CAT, and MDA activities were monitored by measuring absorbance at 550 nm, 420 nm, 405 nm, and 530 nm, respectively.

Metabolites analysis

After 15 days of treatment, lettuce leaves were harvested and immediately frozen in liquid nitrogen. A methanol-water solution (1:1, V/V) was used to extract metabolites. Then, the mixture was ground for 4 min and ultrasonicated for 5 min. After the centrifugation for 15 min at 12,000 r/min, 250 µL of supernatant was collected and dried. To redissolve the residue, 50 µL of a methanol-water solution (1:1, V/V) was used and centrifuged at 12,000 r/min for 15 min. The supernatant was used for metabolic analysis.

The metabolites were analyzed using LC-MS/MS, which consists of a Vanquish HPLC and a Orbitrap Exploris 120 mass spectrometer. Metabolites were separated by a Waters ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) with a mobile phase including 5 mmol/L aqueous ammonium acetate and ACN. The detailed MS parameters are as follows: sheath gas flow rate: 50 Arb, aux gas flow rate: 15 Arb, capillary temperature: 320℃, full MS resolution: 60,000, MS/MS resolution: 15,000, collision energy: 10/30/60, spray V voltage: ±3.4 kV.

Dietary intake risk of colistin in lettuce to local residents

The potential risk from consuming colistin-contaminated lettuce was assessed using the Estimated Daily Intake (EDI), calculated as follows [20]:

Where EDI is expressed in µg/(kg bw d); C (µg/kg) represents the concentration of colistin; CI (kg/day) is the daily intake of vegetable, set at 0.5 kg/day according to the Dietary Guidelines for Chinese Residents [21], and BW is the average human body weight (60 kg).

The hazard quotient (HQ) is calculated as the ratio of EDI to acceptable daily intake (ADI), serving as an indicator of the health risk associated with chronic dietary exposure. The ADI value of colistin is 7 µg/(kg bw d) according to the recommendation of FAO [22]. An HQ value less than 1 indicates an acceptable health risk with no expected long-term effects, whereas an HQ greater than 1 suggests a potential health risk [23].

Statistical analysis

All experiments were performed in quintuplicate, and data were presented as mean ± standard deviation (SD). Significant differences were identified by one-way analysis of variance (ANOVA) followed by Duncan’s tests between the blank group and treated groups using the IBM SPSS 22 software. All figures were produced using Origin 2021.

Results

Analysis of colistin residue in lettuce by LC-MS/MS coupled with DSPE method

Colistin is a multi-component antibiotic with its main components of colistin A and colistin B, so the recovery in this study is the sum of the recoveries of colistin A and colistin B. In this paper, four extracts, including ACN/MeOH-1% FA aqueous solution and different proportions of MeOH-0.1% FA aqueous solutions, were investigated. As shown in Fig. S1, it is obvious that a rich aqueous phase is conducive to increasing the extraction efficiency of colistin. Furthermore, with the increase of formic acid, the recovery of colistin increased remarkably.

According to the guidelines outlined in Commission Decision 2002/657/EC, validation procedures must be conducted regularly to ensure that a new method meets fit-for-purpose criteria. Matrix-matched standard solutions were prepared to generate calibration curves. For colistin, good linearity was observed at spiked concentrations of 10∼400 µg/kg, with correlation values better than 0.99. Table 1 summarizes the recoveries together with the relative standard deviation (RSD) that corresponds to them. The average recoveries of colistin at the spiking doses of 10, 30, 100, and 300 µg/kg were found to be between 73.3% and 91.1%, with RSDs less than 7.7%. These results are within the permitted ranges for drug residue analysis. Figure 1 shows typical chromatograms obtained from spiked lettuce at 10 µg/kg. The limit of detection (LOD) and LOQ were 5 µg/kg and 10 µg/kg, respectively.

Table 1.

Recovery and precision of colistin in lettuce

Analyze compounds	Concentration(µg/kg)	Intra-day recovery (RSD,%,n=6)			Inter-day recovery(RSD,%, n = 18)
		First	Second	Third
Colistin A	10	76.5(6.0)	74.4(6.1)	71.9(9.4)	74.3(7.7)
	30	79.5(5.9)	78.2(5.6)	78.7(8.8)	78.8(6.9)
	100	86.6(5.7)	84.1(5.2)	83.4(7.4)	84.7(6.4)
	300	89.7(4.0)	88.5(3.1)	87.6(6.3)	88.6(4.8)
Colistin B	10	73.1(7.7)	69.5(5.3)	77.4(5.2)	73.3(7.6)
	30	78.1(8.4)	76.2(5.7)	81.4(3.8)	78.6(6.8)
	100	82.8(6.7)	86.6(5.0)	85.7(6.1)	85.0(6.2)
	300	92.2(3.5)	91.2(2.7)	90.1(3.0)	91.1(3.2)

Fig. 1.

Typical chromatogram of lettuce added with 10 µg/kg colistin (A−1 and B−1) and blank lettuce matrix (A−2 and B−2): CSA, colistin A. CSB, colistin B

The applicability and reliability of the method were validated using positive control lettuce samples and 50 samples collected from local supermarkets. In the 300 µg/kg colistin treatment group, the total colistin residue is 90.5 µg/kg, and in the 30 µg/kg group it was 6.4 µg/kg, but colistin was not detected in the 10 µg/kg group (Fig. S2). No colistin residues were detected in any of the real lettuce samples.

Effects of colistin on seed germination, root length, shoot length and chlorophyll content of lettuce

Figure 2A shows the lettuce germination rate. Seed germination increased with increasing concentrations of colistin, but no significant differences were observed. Low concentrations of colistin have minimal effect on the germination energy of lettuce seeds, whereas medium and high concentrations significantly enhance germination energy (Fig. 2B). Colistin inhibited the growth of lettuce in a dose-dependent manner (Fig. 2C and D). The root and shoot lengths in the control group were significantly longer than those of every colistin treatment group. The root lengths of the 10, 30, and 300 µg/mL colistin treatments were 47.1%, 70.6%, and 99.2% shorter than those of the control group, respectively. The length of the shoots in the colistin treatments was 83.1%, 55.9%, and 44.2% shorter than in the control, respectively.

Fig. 2.

Effects of colistin on germination rate (A), germination energy (B), and root and shoot length (C and D) of lettuce. Different letters indicate significant differences when P<0.05

Ultrastructural changes

As shown in Fig. 3, transmission electron microscopy (TEM) analysis revealed significant damage in chloroplasts and mitochondria of lettuce leaf cells under colistin exposure. In the control group, chloroplasts exhibited a characteristic elliptical morphology (Fig. 3A-1) with well-organized grana stacks and well-ordered stromal thylakoid vesicles; mitochondria had well-defined cristae structures and a homogeneous matrix density (Fig. 3B-1), indicating normal photosynthetic functionality and cellular respiration. However, colistin was able to induce significant damage to chloroplasts, including membrane disruption, disorganized stromal lamellae, and loss of organelle boundary integrity (Fig. 3A-2). Furthermore, mitochondria exhibited characteristic pathological changes of organelle swelling, cristae fragmentation, and matrix density reduction (Fig. 3B-2).

Fig. 3.

Transmission electron microscope (TEM) images of chloroplasts A and mitochondria B in control group A-1 and B-1 and 300 μg/kg colistin treated group A-2 and B-2 CP, chloroplast. Mi, mitochondria. LD, lipid droplets. SG, starch granule. V, vacuole

Changes of SOD, POD, CAT, MDA and chlorophyll levels

The effects of colistin on the antioxidant enzymes (SOD, POD, and CAT) and MDA are shown in Fig. 4. Compared with the control, the 10, 30, and 300 µg/kg colistin treatment groups demonstrated 26.4%, 29.0%, and 37.7% higher SOD activity, respectively (Fig. 4A). The POD values increased by 53.0%, 58.8%, and 62.2% in colistin treatment groups, respectively, indicating that colistin can trigger oxidant stress (Fig. 4B). The CAT activity in the 30 and 300 µg/kg colistin treatment groups was 24.5% and 45.9% lower than the control group, respectively (Fig. 4C). However, there was no significant difference in 10 µg/kg colistin treatment compared with the control group. Compared with the control, MDA content in 10, 30, and 300 µg/kg colistin treatment groups increased by 36.3%, 44.8%, and 54.7%, respectively (Fig. 4D). The chlorophyll content in the colistin treatment group was significantly lower than that in the control group (Fig. 4E). The plants treated with colistin exhibited declines in their chlorophyll content of 16.1%, 19.4%, and 25.4%, in comparison to the control group.

Fig. 4.

Effects of different concentrations of colistin on superoxide dismutase (SOD, A), peroxidase (POD, B), catalase (CAT, C), malondialdehyde (MDA, D) and chlorophyll content (E). Different letters indicate significant differences when P< 0.05

Metabolomics analysis

Metabolomics provides a comprehensive approach for qualitative and quantitative characterization of metabolic profiles in organisms subjected to various biotic or abiotic stresses. To investigate the metabolic responses of lettuce under colistin stress, an untargeted metabolomics analysis was performed employing ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). A total of 1389 metabolites were identified and quantified in this study, offering a systematic overview of the metabolic alterations in lettuce under colistin stress.

Initial principal component analysis (PCA) demonstrated minimal separation trends among groups (Fig. S3A). To further analyze the differences and find differential metabolites, we performed orthogonal partial least squares–discriminant analysis (OPLS-DA). The OPLS-DA score revealed a distinct separation between the control group and the colistin group(Fig. S3B). The established model demonstrated excellent explanatory power (R²Y = 0.992) and stable predictive capability (Q2 = 0.515) (Figure S3C) (Fig. S3C). These results suggested that the metabolic program of lettuce was significantly changed under colistin stress.

A volcano plot analysis visualized the differential abundance and statistical significance of metabolites between two groups. With the threshold criteria (VIP > 1 and p < 0.05), 57 significantly altered metabolites were identified in lettuce under 300 µg/mL colistin stress, including 27 upregulated and 30 downregulated metabolic features (Fig. 5A). The heatmap revealed a distinct separation between the colistin-treated and control groups (Fig. 5B), demonstrating that colistin can alter metabolites significantly.

Fig. 5.

Metabolome alteration of lettuce leaves under 300 µg/kg colistin treatment: volcano plots (A), heat map showing the changes of metabolites in lettuce (B), the histogram showing the enrichment analysis of differential metabolic compound pathways of lettuce (C), differential abundance score plot of differential metabolic pathways, the depth of the color of the line segment and the dot is proportional to the DA score (D)

Metabolic pathway analysis was performed by mapping differential metabolites to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.kegg.jp), followed by systematic evaluation and functional categorization. 14 enriched metabolic pathways (P < 0.05) affected by colistin in lettuce were screened (Fig. 5C and D). Colistin significantly disturbed multiple biological processes in lettuce, including lipid metabolism, cofactor and vitamin metabolism, and secondary metabolite biosynthesis, particularly affecting lipid metabolism. Colistin seriously disturbed the arachidonic acid metabolic pathway of lettuce, with three key metabolites of 5,6-DHET, phosphatidylcholine (PC (18:1(11Z)/15:0)), and prostaglandin G2, exhibiting significant alterations. Among these, PC (18:1(11Z)/15:0) was the most enriched differential metabolite.

Dietary intake risk of colistin in lettuce to local residents

In this study, 50 lettuce samples were collected from local supermarkets, and colistin was not detected in any of them. Based on the residue levels in these real samples, the hazard quotient (HQ) was calculated as 0, indicating a low dietary risk of colistin exposure to local residents. In the positive control lettuce samples, no colistin was detected in the group treated with 10 µg/kg. However, colistin residues of 6.4 µg/kg and 90.5 µg/kg were detected in lettuce exposed to 30 µg/kg and 300 µg/kg colistin, respectively. These residues can exert toxic effects on lettuce growth and development. Furthermore, a significant concern is the potential for bacterial resistance arising from residual colistin in lettuce entering the human body through the food chain.

Discussion

The development of a rapid LC-MS/MS method to detect colistin in lettuce

Due to the water solubility and high polarity of colistin, different ratios of MeOH-formic acid aqueous solutions are usually used to extract colistin from feed, animal tissue, and egg, while the extract of the target compound from vegetables has not been reported. It was reported that the low pH environment can effectively block the interaction between colistin and proteins, facilitating the liberation of colistin [24]. Therefore, MeOH-1% FA aqueous solution (1:5) was used to extract colistin with its recovery higher than 90%.

Reported literature commonly involved a SPE strategy to purify tissue or egg matrices. However, with high percentages of sugar, protein, and fiber existing in lettuce, the extract solution’s high viscosity and more precipitates made it difficult to pass through the SPE cartridge. Poor recovery (lower than 40%) was obtained with the SPE procedure. Accordingly, a dilution combined with a DSPE protocol was performed in this study. The DSPE is a flexible and eco-friendly method, which is commonly used in sample purification with less loss of analytes.

Colistin inhibits the growth of lettuce

Typically, the growth cycle of fast-growing lettuce spans 4 to 8 weeks, encompassing seed germination, seedling development, and maturity stages. Based on literature and national standards, seed germination was analyzed on day 3, and germination rate was assessed on day 7. The 15-day time point, corresponding to the seedling stage, was selected for microscopic observation of lettuce organelles, chlorophyll content measurement, oxidative stress indicator assessment, and metabolomics analysis. During this stage, numerous secondary metabolites and antioxidant compounds are synthesized and accumulated, with their dynamic changes closely associated with plant stress responses and growth regulation [25]. Moreover, seedlings typically exhibit higher absorption rates of pollutants and nutrients compared to mature plants [26].

The first phase of the plant life cycle, seed germination, is susceptible to external factors. It has been reported that high concentrations of antibiotics significantly decreased seed germination, while low concentrations had no effect [14]. However, another study found that low concentrations of antibiotics promoted seed germination [10]. The germination rate strongly correlates with the type of antibiotic, the treatment dose, and the plant species. The increase of seed germination in the present study may be due to emergency mobilization of hormone or protein in lettuce seeds in an attempt to counteract harmful environmental variables [27]. Much literature has confirmed that antimicrobials inhibit the development of roots and shoots and that this inhibition is positively connected with drug concentration. The findings of the present study agree with this literature. The suppression of plant growth factors (e.g., growth hormone, cytokinin, and ethylene) under drug stress may be the cause of the inhibition of root growth.

Previous studies have shown that most antibiotics (such as oxytetracycline, ofloxacin, and sulfonamides) reduce the amount of chlorophyll in plants [28]. The higher the drug concentration and the longer the treated time, the lower the chlorophyll content in the leaves, similar to the results of the present study. Furthermore, studies have reported a strong correlation between nitrogen and chlorophyll, with leaves containing 80% of nitrogen in the chloroplast [29]. Nitrogen can stimulate the development of plant roots, shoots, and leaves. Thus, one of the reasons for the suppression of root and shoot lengths in lettuce under colistin stress may be that drop in nitrogen content.

Colistin damages the chloroplasts and mitochondria of lettuce

Chloroplasts, as essential organelles in plants, are critically involved in maintaining photosynthetic efficiency. Many studies have demonstrated that both pigment degradation and structural damage to chloroplasts can significantly impair photosynthetic performance. For instance, under ketoprofen stress, rice seedlings’ chloroplast structure suffered damage, leading to a concentration-dependent decrease in photosynthetic pigment content [30]. Consistent with these findings, the dramatical reduction in pigment content under colistin stress was likely attributable to chloroplast damage. Changes in mitochondrial membrane permeability can trigger the release of ions and solutes, resulting in mitochondrial swelling and the rupture of the outer mitochondrial membrane. Lipid droplets participate in various cellular processes such as stress response mechanisms, pathogen defense systems, and hormonal metabolism regulation [31]. The lipid droplets in the intercellular spaces of lettuce under colistin stress increased suggesting that colistin may disrupt intracellular lipid homeostasis.

Colistin enhances oxidative stress damage of lettuce

The antioxidant defense system can prevent the detrimental effects of ROS and lipid peroxidation. An essential part of the antioxidant defense system is the SOD, which scavenges O²⁻ through converting them into H₂O₂ and O₂. Numerous studies have indicated that oxidative stress can significantly increase the activity of SOD [32]. Previous studies have demonstrated that ciprofloxacin and erythromycin increase POD activity [33], aligning with our findings. CAT is one of the important enzymes for scavenging ROS in plants. It could reduce the quantity of harmful H₂O₂ by converting it to H₂O and O₂, thereby relieving damage to plants. A previous study reported that SOD activity rises and CAT activity falls under fluoroquinolone stress [34]. However, another study has confirmed that tetracycline can decrease SOD activity and increase CAT activity [35]. These contradictory findings demonstrated that the antioxidant defense system is dynamic and varies over time. Plants constantly modify enzyme activities to precisely control ROS levels in response to environmental stress [11]. MDA is the decomposition product of polyunsaturated fatty acids and an indirect indicator of cell injury and lipid peroxidation. Under drug stress, the massive accumulation of ROS could increase the production of MDA. In this study, the POD and SOD content was increased and the CAT content was decreased, showing that under colistin stress, a large production of reactive oxygen species in lettuce, and the plant body cannot provide enough catalase to resist oxidative stress, leading to oxidative damage. The level of MDA, a lipid peroxide, also serves as an indirect indicator of the extent of plant oxidative damage [36]. The MDA content showed that lipids were affected, which was consistent with the metabolic pathway in which arachidonic acid metabolism was affected. It has been found that mucilage is lipophilic, so we hypothesize that mucilage may act on lipid-related pathways to disrupt the plasma membrane of lettuce, affecting its growth.

Colistin disturbs the phospholipid-arachidonic acid metabolic pathway of lettuce

As fundamental structural components of biological membranes, lipids play crucial roles in stress responses. Plasma membrane receptors initially perceive abiotic stresses, such as drought, salinity, temperature extremes, oxidative stress, and chemical toxicity, which negatively impact plant growth. For instance, lipids can facilitate cold stress tolerance in rice [37]. When polyvinyl chloride microplastic particles adhered to the plant, lipid synthesis was stimulated while the production of lipid-related metabolites was inhibited [38]. In this study, the metabolic pathways of arachidonic acid, linoleic acid, glycerophospholipids, α-linolenic acid, and sphingolipids in lipid metabolism were significantly altered in the colistin stress group compared to the control group. Among them, arachidonic acid is the most affected metabolic pathway. Arachidonic acid belongs to the n-6 series of polyunsaturated fatty acids (PUFA) and is involved in the synthesis of unsaturated fatty acids. PUFA participates in plant membrane formation and is also related to plant stress tolerance [39]. Arachidonic acid-induced immunity has been experimentally demonstrated in various plant species, including tobacco, foxtail millet, and potato. Numerous studies have shown that application of arachidonic acid significantly enhances the activities of guaiacol peroxidase, polyphenol oxidase, and proteolytic enzymes in lettuce leaves, thereby increasing resistance to Botrytis cinerea infection [40].

Phosphatidylcholine, as a predominant phospholipid constituent, plays crucial roles in maintaining membrane integrity across various cellular compartments, including plasma membranes, endoplasmic reticulum, and chloroplast outer membranes [41]. The reduction of phospholipid content in the outer membrane of chloroplasts may reduce photosynthetic efficiency, potentially explaining the decreased chlorophyll content in lettuce under colistin stress. The significant downregulation of 5,6-DHET and prostaglandin G2 indicated potential inhibition of their biosynthetic enzymes cytochrome P450 (CYP450) and cyclooxygenase by colistin. CYP450 is one of the largest gene families in plants and plays an important role in numerous physiological processes, including hormone biosynthesis and signal transduction [42]. Cyclooxygenase serves as a key enzymatic regulator in the biosynthesis of lipid-derived signaling molecules that mediate plant defense responses against pathogen invasion. Furthermore, under colistin stress, a significant upregulation of the differential metabolite 11(R)-HETE was observed, which is linked to the lipoxygenase (LOX)-mediated oxidative pathway. LOX is a key enzymatic regulator in the catalysis of polyunsaturated fatty acid peroxidation, which is a critical process in oxidative stress responses. It was reported that exogenous material (e.g., Hg) can trigger the activation of LOX, further causing the excessive generation of ROS and membrane lipid peroxidation, which ultimately damage the integrity of cellular membranes and ultrastructural alterations in rice root tissues [43]. These findings underscore the pivotal role of LOX-mediated lipid peroxidation in xenobiotic-induced phytotoxicity. Therefore, colistin induced structural damage to lipid membranes and chloroplast stroma in lettuce. These impairments reduced photosynthetic efficiency and energy metabolism, consequently inhibiting lettuce growth and development, as evidenced by increased germination energy, shorter roots, and decreased chlorophyll content. The toxicity mechanism of colistin in lettuce is likely associated with disruption of the phospholipid-arachidonic acid metabolic pathway. This disruption leads to an imbalance in bioactive lipid mediators—characterized by upregulation of 11(R)-HETE and downregulation of 5,6-DHET and prostaglandin G2—which results in abnormal oxidative stress responses, including increased MDA levels, elevated POD and SOD activities, decreased CAT activity, and ultimately triggers a burst of ROS (Fig. 6).

Fig. 6.

The phytotoxic mechanism of colistin pressure on lettuce

Due to the lack of established residue detection methods, there are no published reports on colistin residues in vegetables, making it impossible to obtain such data from the literature. In this study, 50 lettuce samples purchased from local supermarkets were analyzed, and no colistin residues were detected (HQ = 0), indicating a low dietary risk of colistin exposure for local residents. The absence of colistin residues in real lettuce samples may be attributed to the strict regulation limiting colistin use as an animal feed additive in China or to the degradation of colistin during lettuce growth. Previous studies have reported that colistin degradation in soil, originating from animal feces, is closely related to soil properties; for example, colistin at concentrations ranging from 10 to 40 µg/g in sandy loam soil can be completely degraded within 65.75 to 148.25 days [44]. In the positive control lettuce samples, no colistin was detected in the low-dose treatment group, whereas residues of 6.4 µg/kg and 90.5 µg/kg were detected in the medium- and high-dose treatment groups, respectively. If these positive samples undergo an adequate withdrawal period prior to marketing, colistin residues can degrade to undetectable levels. Therefore, the risk of colistin exposure to humans through dietary intake of lettuce is very low. Nonetheless, despite the low dietary risk, the phytotoxic effects of colistin on plant growth and the potential for antimicrobial resistance arising from prolonged exposure to colistin-contaminated products should not be ignored. The synergistic effects of colistin accumulation in vegetables combined with other antimicrobial agents or environmental contaminants, such as heavy metals and microplastics, may potentially exacerbate long-term health risks [45].

The application of animal manure containing colistin varies significantly across countries [46]. Colistin concentrations in swine feces have been reported to reach 48.6 µg/g in Belgium [47], while in China, levels range from below the limit of quantification to 17.4 µg/g [4]. Due to regional differences in colistin usage, sampling ranges, and sample sizes, further research is necessary for comprehensive risk assessment. Additionally, the emergence of colistin-resistant bacteria and resistance genes has led to increased colistin dosages for disease treatment. More colistin treatment results in greater environmental release of residues, potentially promoting the proliferation of drug-resistant bacteria. The potential for antibiotic resistance is not addressed in the dietary exposure assessment based on the ADI used in this study, which primarily evaluates toxicological effects of chronic consumption such as allergies and nephrotoxicity. According to Environmental Health Criteria 240, two critical factors should be considered when establishing a microbiological ADI to assess antibiotic resistance risks: (1) compromised barriers to bacterial colonization and (2) an increase in resistant bacterial populations. Unfortunately, no microbiological ADI for antibiotics is currently available.

Consumption of contaminated raw vegetables may facilitate the transmission of antimicrobial-resistant bacteria or resistance genes to humans, potentially leading to foodborne disease outbreaks. Currently, the colistin resistance gene (MCR-1) has been has been detected in various vegetables (lettuce, tomatoes, Chinese cabbage, green peppers, cucumbers, carrots, etc.) in many countries such as Portugal, Switzerland, China, India, and South Korea (Table 2). The detection rate of MCR-1 in lettuce is 4.3%. However, these studies did not report the determiantion of colistin residues in the vegetables. Therefore, the extraction and detection method for colistin in lettuce developed in this study, when combined with MCR-1 detection, will contribute to reveal the reasons behind the widespread dissemination of colistin resistance in the chain of “animal-environment-vegetable-table.”

Table 2.

Colistin resistance gene (MCR-1) detected in various vegetables

Origin	Species	MIC of colistin (µg/mL)	Other resistance phenotypes	Country	Reference
Lettuce	Escherichia coli	/	AMP, N, GEN, C	Portugal	[48]
Lettuce	M.morganii	/	AMP, Ac, N, GEN, C
Strawberry	K. pneumoniae	/	AMP, Ac, CTX, Cr, N CIP, S
Basil leaves	Escherichia coli	6	/	Vietnam	[49]
Cha-om	Escherichia coli		/	Thailand
Lettuce	Raoultella ornithinolytica	4–8	/	China (Guangzhou)	[50]
Lettuce	Escherichia coli	4–8	/	China (Guangzhou)
Tomato	Escherichia coli	8	/	China (Guangzhou)
Vegetable	Klebsiella spp	16–256	/	India	[51]
Cucumber	Escherichia coli	8	AMP, NAL, CIP, ENR, LEV, KAN, GEN, TET, DOX, FOS	China (Beijing, Shanghai, Shandong)	[52]
Tomato	Escherichia coli	8	AMP, NAL, CIP, ENR, GEN, TET, DOX*
Leaf rape	Escherichia coli	8	AMP, NAL, CIP, ENR, LEV, KAN, GEN, TET, DOX
Carrot	Escherichia coli	8	AMP, CTX, CTF, NAL, ENR, KAN, GEN, TET, DOX, FOS
Romaine lettuce	Escherichia coli	8	AMP, CTX, CTF, NAL, KAN, GEN
Green pepper	Escherichia coli	8	AMP, KAM, GEN, TET, DOX
Curly endive	Escherichia coli	8	AMP, NAL, ENR, LEV, GEN, TET, DOX*
Cucumber	Escherichia coli	4	AMP, NAL, CIP, ENR, LEV, GEN, TET, DOX
Pak choi	Escherichia coli	8	AMP, CTX, CTF, NAL, CIP, ENR, LEV, KAN, GEN, TET, DOX
Leaf lettuce	Escherichia coli	8	AMP, CTX, CTF, NAL, CIP, ENR, LEV, KAN, GEN, AMK, TET, DOX, FOS
Spinach	Escherichia coli	4	AMP, NAL, ENR*, KAN*, GEN, TET, DOX*
Green pepper	Enterobacter cloacae	/	AMP, CTX, CTF, NAL, CIP, ENR, LEV, KAN, GEN, TET, DOX, FOS
Lettuce	Escherichia coli	8	/	South Korea	[53]

MICs Minimal inhibit concentration. Abbreviations for antibacterial agents or antibiotics, AMP Ampicillin, Ac Amoxicillin with clavulanic, CTX Cefotaxime, Cr Ceftazidime, CTF Ceftiofur, NAL Nalidixic acid, CIP Ciprofloxacin, C Chloramphenicol, N Nalidixic, S Trimethoprim/sulfamethoxazole, ENR Enrofloxacin, LEV Levofloxacin, KAN kanamycin, GEN Gentamicin, AMK Amikacin, TET Tetracycline, DOX Doxycycline, FOS Fosfomycin

Conclusion

In this study, a rapid, environmentally friendly, and sensitive LC-MS/MS method was developed for the detection of colistin residues in lettuce. Method validation confirmed the sensitivity and reliability of the approach. Exposure to colistin induced significant oxidative stress in lettuce, evidenced by increased levels of ROS, MDA, and SOD activity, alongside decreased CAT activity and chlorophyll content. The TEM results revealed structural damage to chloroplasts and mitochondria. Metabolomic analysis demonstrated that colistin disrupted the phospholipid-arachidonic acid metabolic pathway, causing significant alterations in metabolites such as 5,6-DHET, phosphatidylcholine, and prostaglandin G2. The observed oxidative stress and organelle damage were closely associated with the imbalance metabolism, ultimately inhibiting lettuce growth. Although the dietary risk of colistin exposure through lettuce consumption appears low, its potential to induce antimicrobial resistance and synergistic toxicity with other environmental contaminants merits further investigation.

Abbreviations

MCR-1

The colistin resistance gene

LC-MS/MS

Liquid chromatography tandem mass spectrometry

MDA

Malondialdehyde

POD

Peroxidase

SOD

Superoxide dismutase

CAT

Catalase

MDA

Malondialdehyde

HQ

Hazard quotient

MDR

Multidrug-resistant

ROS

Reactive oxygen species

SMZ

Sulfamethoxazole

EMP

Glycolytic pathway

HPLC

High performance liquid chromatography

FA

Formic acid

MeOH

Methanol

ACN

Acetonitrile

PSA

Primary secondary amine

DSPE

Dispersive solid-phase extraction

TEM

Transmission electron microscopy

SD

Mean ± standard deviation

ANOVA

Analysis of variance

RSD

The relative standard deviation

LOD

Limit of detection

LOQ

Limit of quantitation

UPLC-MS/MS

Ultra-performance liquid chromatography coupled with tandem mass spectrometry

PCA

Initial principal component analysis

OPLS-DA

Orthogonal partial least squares–discriminant analysis

KEGG

Kyoto encyclopedia of genes and genomes

SPE

Solid phase extraction

PUFA

Polyunsaturated fatty acids

CYP450

Cytochrome P450

LOX

Lipoxygenase

GE

Germination energy

GR

Germination rate

EDI

Estimated daily intake

ADI

Acceptable daily intake

Authors’ contributions

X.S. and J.Y. designed the experiments, provided financial support, and revised the manuscript. S.P and H.T. conducted the experiments and wrote the first draft of the manuscript. Y.C., H.Z., S.L., and D.O. conducted the experiments and collected the data. All authors reviewed the manuscript.

Funding

This work was supported by Guizhou Provincial Basic Research Program (Natural Science) (No. QKHJC-ZK-2023-109; QKHJC-ZK-2022-129), National Natural Science Foundation of China (32202854), Special Funds of the Natural Science Foundation of Guizhou University ([2020]25), Graduate Research Fund Project of Guizhou Province (2024YJSKYJJ124).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.