Response of Nicandra physalodes (Linn.) Gaertn. and Its Rhizospheric Organisms to the Selective Pressures of High-Concentration Oxytetracycline, Ciprofloxacin, and Tobramycin

Abstract

Antibiotics play an essential role in the treatment of infectious diseases in humans and animals. Despite their benefits, the release of an increasing amount of various antibiotics into the environment poses a potential threat to plants, soils, animals, and microorganisms. Here, an outdoor pot experiment was conducted to preliminarily evaluate high concentrations of three antibiotics (oxytetracycline, ciprofloxacin, and tobramycin) on Nicandra physalodes (Linn.) Gaertn. and its rhizospheric organisms. The results showed that the three antibiotics had different effects on the growth behavior (germination uniformity, average growing height gain per week, and thousand seed weight) and biomass (aboveground biomass and belowground biomass) of Nicandra physalodes (Linn.) Gaertn. After adding high concentrations of the three antibiotics to the soil of each test group, the earthworm extract in group A (oxytetracycline) significantly increased the coagulation parameters prothrombin time (PT) and thrombin time (TT). The PT significantly decreased (p < 0.01), while the TT was not affected in group B (ciprofloxacin). For group C (tobramycin), the TT significantly decreased (p < 0.01) and the PT was not affected compared to the CK group (control group). The application of the three antibiotics caused alterations in the general activity of enzymes, such as soil sucrase (SC), urease (UE), acid phosphatase (ACP), alkaline phosphatase (AKP), and nitrate reductase (NR). Different antibiotic groups influenced the rhizosphere bacterial diversity and community structure of Nicandra physalodes (Linn.) Gaertn. At the phylum level, Ignavibacteriae was only found in group C, and Parcubacteria and Ignavibacteriae were not present in the CK group. At the genus level, Parcubacteria_norank was not present in the CK group, and BSV40_norank was found in group C. Ultimately, the results suggested that high concentrations of oxytetracycline, ciprofloxacin, and tobramycin could affect the growth behavior and biomass of Nicandra physalodes (Linn.) Gaertn. and alter its rhizobacterial community structure, providing the scientific basis for the rational use of antibiotics in animal husbandry and veterinary science.

1. Introduction

Antibiotics play an important role in the treatment of infectious diseases in humans and animals. However, the release of an increasing amount of various antibiotics into the environment poses potential threats to plants, soils, animals, and microorganisms. Meanwhile, their potential adverse impact, the emergence of antibiotic resistance genes (ARGs) among microbial populations in soil, and the risk of animal and human infection are of great concern [1]. Apart from the selection of antibiotic-resistant microorganisms and ARGs in soils, antibiotics may also affect functional, structural, and genetic diversity [2]. Many reports have detected relatively high residual levels of fluoroquinolones, tetracyclines, and aminoglycosides in the soil, and their residue, pollution, migration, and degradation in the soil were also thoroughly discussed [3,4,5]. Unfortunately, there is still a lack of systematic and comprehensive reports on the absorption, accumulation, migration, and toxic effects of their residue on the biological communities of Nicandra physalode (Linn.) Gaertn. and its rhizospheric organisms.

Nicandra physalode (Linn.) Gaertn. is an alien plant from southwest China and is commonly used for the treatment of sedative, expectorant, and fever relief based on its rheological properties and chemical compositions around the Qinghai–Tibet plateau [6]. However, for such a medicinal plant, the residual effect of veterinary antibiotics on its growth behavior and rhizospheric organisms is currently unknown. Here, an outdoor pot experiment was conducted to preliminarily evaluate high concentrations of three antibiotics (oxytetracycline, ciprofloxacin, and tobramycin) on Nicandra physalodes (Linn.) Gaertn. and its rhizospheric organisms. The study goals were to (1) evaluate the effect of antibiotics on the growth behavior and biomass of Nicandra physalodes (Linn.) Gaertn, (2) compare the blood coagulation characteristics of earthworm extracts under antibiotic selective pressures, and (3) evaluate the contributions of the antibiotics in structuring the rhizosphere bacterial communities of Nicandra physalodes (Linn.) Gaertn. The results could provide the scientific basis for the absorption, accumulation, and migration of antibiotics in Nicandra physalodes (Linn.) Gaertn. and other medicinal plants in the Qinghai–Tibet plateau.

2. Material and Methods

2.1. Plants, Earthworms, Antibiotics, and Reagents

Fresh ripe seeds of Nicandra physalodes (Linn.) Gaertn. were collected from the Hailuogou Scenic Area (101°49′~102°10′ E, 29°20′~29°55′ N, approx. 1600 m above sea level), Moxi Town, Luding County, Sichuan province, China on 20 October 2017. Healthy earthworms (Eisenia foetida) were kindly provided by an earthworm breeding factory in Shuangliu District, Chengdu, Sichuan province. The seed and voucher specimens were deposited at the Laboratory of Veterinary Pharmacology and Toxicity of Southwest Minzu University. Oxytetracycline (purity ≥ 95%), ciprofloxacin (purity ≥ 98%), and tobramycin (purity ≥ 98%) were purchased from Shanghai Yuanye Bio-technology Co., Ltd. (Shanghai, China). The commercial kits for the soil enzyme activities, including urease (UE, LOT NO. BC0120), sucrase (SC, LOT NO. BC0240), acid phosphatase (ACP, LOT NO. BC0145), alkaline phosphatase (AKP, LOT NO. BC0280), and nitrate reductase (NR, LOT NO. BC0080), were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The molecular biology reagents for DNA extraction and library construction were purchased from Axygen. The 16s rRNA sequencing was completed by Annoroad Gene Technology Co., Ltd. (Beijing, China). All the other biochemical analysis reagents used in this study were of analytical grade.

2.2. Soil Treatment, Earthworm Stocking, and Seed Sowing

The black sandy soil samples were collected from the No.3 Camp in the Hailuogou Scenic Area (approx. 3200 m above sea level), and the three antibiotics (oxytetracycline, ciprofloxacin and tobramycin) were not detected by the high-performance liquid chromatography method (data not published). The main chemical properties were analyzed according to the Zhang et al. report [7]: organic matter, 181.19 g/kg; total nitrogen, 6.81 g/kg; total phosphorus, 0.84 g/kg; total potassium, 29.1 g/kg, and a pH of 6.79.

The soil samples were air-dried, crushed, passed through an 80-mesh screen, and fully mixed with the three antibiotics. The dosages were calculated according to the preset concentrations. The soil samples containing high concentrations of oxytetracycline (25 g/kg), ciprofloxacin (30 g/kg), and tobramycin (20 g/kg) were named group A, group B, and group C, respectively, and the control group without antibiotics was named group CK.

The outdoor pot experiment was conducted as follows. Three layers of soil were added to the enamel pots and an equivalent number of healthy earthworms were introduced after the first two layers were completed. A total of 300 seeds of Nicandra physalodes (Linn.) Gaertn. was placed in each pot using the dibble seeding method (the germination percentage was above 95% and the thousand seed weight was 2.99 ± 0.02 g in pre-testing) after adding the third loose layer of soil. After sowing, the remaining soil was added evenly and a shower was used to moisten the soil with deionized water. The following experiment was carried out under natural conditions to keep the soil moist.

2.3. Effects of Antibiotics on the Growth Behavior and Biomass of Nicandra physalodes (Linn.) Gaertn

This experiment was carried out from 2 May to 23 November 2018. The growth behavior of Nicandra physalodes (Linn.) Gaertn., such as the germination uniformity, average growing height gain per week, and thousand seed weights, were recorded at a fixed sampling point. To evaluate the aboveground and belowground biomass, the plant samples were oven-dried at 70 °C until they achieved a constant weight, and the dried samples were weighed according to the drying constant weight method [8]. The ripe fruits were harvested after the rhizosphere soil was sampled and the thousand-grain weight was calculated.

2.4. Blood Coagulation Test

For the blood coagulation test, the earthworm extract was prepared according to the reference [9]. Briefly, after the rhizosphere soil samples were collected and the seeds were harvested, the fresh earthworm tissues were added to ultrapure water at a 1:10 ratio (earthworm: water) and initially boiled for at least 0.5 h. Another extract was obtained from the pretreated earthworm. The crude extracts were mixed and filtered before being concentrated at 55 °C via vacuum evaporation. The lyophilized extract was stored at 4 °C for the anticoagulative activities assays in vitro.

The adult mice blood samples were collected from the abdominal aorta and were put into tubes containing a sodium citrate anticoagulant (anticoagulant: blood = 1:9 in volume). The use of blood mice was approved by the Institutional Animal Care and Ethics Committee of Southwest Minzu University. The test tubes were carefully inverted three to five times and centrifuged at 8000 rpm for 3 min. The supernatant was taken for the thrombin time (TT) and prothrombin time (PT) measurements to evaluate their anticoagulation effects within 2 h [10].

2.5. Effects of Antibiotics on Soil Enzyme Activities

Specific enzyme activities are essential to maintain soil quality and are considered to be useful indicators for the response of microorganisms to the stress caused by antibiotics [11]. In this study, the soil sucrase (SC), urease (UE), acid phosphatase (ACP), alkaline phosphatase (AKP), and nitrate reductase (NR) activities were determined and expressed in accordance with the manufacturer’s instructions (Solarbio, China) after removing the gravel from the fresh soil samples.

2.6. Effects of Antibiotics on Nicandra Physalodes (Linn.) Gaertn. Rhizosphere Microorganism Diversity

For the rhizosphere soil sample collection, all the plant roots were collected, and the excess soil was manually shaken from the roots. The soil that was still attached was immediately placed into a sterile plastic bag for the bacterial communities. All the samples were stored in dry ice cooler boxes until the DNA was extracted. The high-throughput sequencing of the V3–V4 variable regions of the 16S rRNA gene of the rhizosphere soil was characterized using a paired-end (PE) Illumina HiSeq platform. The specific test steps included rhizosphere soil DNA extraction, primer adapter design and synthesis, PCR amplification purification, homogenization treatment, PE library construction, and Illumina sequencing, etc. The V3–V4 region was amplified using the universal primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The optimized PCR condition was as follows. The initial denaturation occurred at 95 °C for 5 min followed by 30 cycles of denaturing at 95 °C for 30 s, annealing at 56 °C for 30 s, an extension at 72 °C for 45 s, a single extension at 72 °C for 10 min, and ended at 4 °C. The PCR product was extracted from a 1.5% agarose gel and was purified using a commercial DNA gel extraction kit. Finally, the purified amplicons were pooled in equimolar concentrations and paired-end sequenced on an Illumina MiSeq platform. The raw data were then subjected to a quality control procedure using the Trimmomatic software (Version 0.39) to filter chimeras. The remaining sequences were clustered to generate operational taxonomic units (OTUs) at a 97% similarity level. A representative sequence for each OTU was assigned to a taxonomic level using the Usearch sequence analysis tool (http://drive5.com/uparse/) and accessed on 23 November 2018. The bioinformatics statistical analysis of the rhizosphere soil bacterial community structure was carried out according to the OTUs cluster analysis and the species taxonomy analysis [12,13]. The effect size measurements analysis (LEfSe) was performed for the biomarkers that were determined to be coupled from the linear discriminant analysis (LDA).

2.7. Statistical Analysis

The experimental data analysis was accomplished using the Statistical Product and Service Solutions (SPSS) software (IBM, Version 19.0). A one-way ANOVA was used for the statistical analysis of the differences in the Alpha diversity indices, TT, PT, and soil enzyme activities. The principal coordinates analysis (PCoA) was implemented based on the unweighted UniFrac distance and clustering relationships were produced based on the Bray–Curtis beta diversity distance metrics, respectively. The values of p < 0.05 and p < 0.01 were considered to be statistically significant and extremely distinctly different, respectively.

3. Results and Discussion

3.1. Growth Behavior and Biomass

In the germination test, Nicandra physalodes (Linn.) Gaertn. growing out of the soil surface was regarded as a successful germination. Group CK and group A germinated early on 6 May, and then gradually grew and reached a germination percentage of more than 85%. The germination uniformity was compared on 11 May and the germination uniformity of the three antibiotic groups were quite different (Figure 1a). Group CK germinated greatly and quickly. A total of 287 seeds were germinated and the germination percentage was 95.67%. Group A was the fastest, with 255 seeds germinating (85.0%); Group B had the slowest germination, with only 55 seeds germinating (18.33%); Group C had the middle germination speed, with 101 seeds germinating (33.67%). Before the analysis of the biomass samples, the statistical results of deaths before flowering showed that there was little difference among the three groups. The deaths in group C occurred at the earliest period, and at the latest in Group B. The aboveground biomass of groups A and C had a significant difference, while the belowground biomass of the antibiotic-adding groups had no statistical difference compared to group CK (Figure 1b).

Plant height is an important indicator, and it has an important influence on the crop yield. The treatment of high concentrations of oxytetracycline and tobramycin promoted the growth of Nicandra physalodes (Linn.) Gaertn. and increased the average growing height gain per week, while ciprofloxacin performed an inhibitory effect on the plant height (Figure 1c). Interestingly, high concentrations of oxytetracycline had the opposite effect [14]. We speculated that it was related to the plant species and antibiotic concentrations.

As an important quality factor, the thousand seed weight is a variable trait under the influence of genetic and environmental factors. Figure 1d showed that, statistically, there was no significant effect between the groups. The highest average value of the thousand seed weight was found in group C (3.01 g), while the lowest average value of thousand seed weight was 2.97 g in group B. According to the Lu et al. report [15], we speculated that the changes in the rhizosphere microorganisms had no significant influence on the genes that trigger flowering and stimulate further plant growth.

3.2. Earthworm Extract on the Coagulation Parameters PT and TT

The earthworm extract had several beneficial biological activities, such as antithrombotic and anticoagulant effects in vitro [16,17]. The results showed that after the addition of a high-concentration pressure of antibiotics to the soil of each test group, the earthworm extract performed different response characteristics, which affected the enzyme activity of the earthworm prothrombin and thrombin (Figure 2). Compared to the CK group, the PT and TT significantly increased in group A (p < 0.01). The PT significantly increased (p < 0.01), while the TT was not affected in group B compared to the CK group. For group C, the TT significantly decreased (p < 0.01) and the PT was not affected.

3.3. Soil Enzyme Activities

The enzyme activity indicates the potential for microbial communities to carry out biochemical processes that are essential to maintain soil quality. The specific enzyme activity is considered to be a useful indicator of the response of microorganisms to the stress caused by antibiotics. Any application of antibiotics that affects the growth of soil microorganisms can induce alterations in the general activity of enzymes [18,19]. The application of the three antibiotics affected the growth of soil microorganisms and induced alterations in the general activity of the enzymes, such as SC, UE, NR, ACP, and AKP (Figure 3). The inhibition of AKP was observed in the soil amended with oxytetracycline at the concentration of 25 g/kg soil (p < 0.01). However, this dosage did not affect the SC, UE, NR, and ACP activities, indicating that only AKP was sensitive to oxytetracycline (Figure 3a). Compared to the CK group, the addition of the three antibiotics on the soil NR activity was not significant (Figure 3c). Berg et al. [19] reported that fungi may be a major producer of enzymes and could be responsible for the observed increases in soil enzyme activities. Moreover, the application of the antibiotics led to the adaptation and proliferation of antibiotic-resistant microorganisms. In this study, the high dosage of the three antibiotics affected the soil enzymes due to contamination. We focused on the effect of high concentrations of oxytetracycline, ciprofloxacin, and tobramycin on Nicandra physalodes (Linn.) Gaertn. and its rhizospheric organisms, their metabolites. The primary factors, such as the soil pH and solubility, should be elucidated in our ongoing work. The microorganisms that were sensitive to oxytetracycline, ciprofloxacin, and tobramycin were killed in the presence of high-dosage antibiotic pressures, resulting in the outgrowth of resistant bacteria and fungi. The ratios of the biomass of bacteria/fungi and/or Gram-positive/Gram-negative bacteria were needed for further evaluating the response of microorganisms to the antibiotics not confined in the ongoing study.

3.4. Influence of the Different Antibiotic Groups on Rhizosphere Bacterial Community Diversity

From nutrient cycling to carbon sequestration, soil microorganisms provide a solid base for natural and agricultural ecosystems to function and are highly influenced by plant type, soil type, and environmental conditions. The interrelationships between plant roots and rhizosphere bacteria are especially relevant and affected by plant species, soil parameters, and external factors such as antibiotics [20,21].

After quality filtering, a total of 1,196,499 clean sequences were obtained from all the samples for further analysis. the Good’s coverage was higher than 0.99, and the average amplification fragment length was approx. 433 bp. Furthermore, the stabilized rarefaction curves suggested that the bacterial communities were well characterized with an increased number of sequences (Figure 4a). The Shannon–Wiener curves nearly reached a plateau, indicating that the sample size was reasonable for the data analysis (Figure 4b). There were 1047, 1133, 1203, and 740 OTUs in all the replicates of group A, group B, group C, and group CK, respectively, and the OTUs shared by all the replicates of group A, group B, group C, and group CK were 890, 920, 999, and 572, respectively (

Table 1. Alpha diversity indices of the bacterial communities in the rhizosphere soils of Nicandra physalodes (Linn.) Gaertn.

Groups	OTUs (Total in the All Replicates/Shared by the All Replicates)	OTUs Richness (X ± SD)	Ace	Chao1	Shannon	Simpson
CK	740/572	572.25 ± 12.26	655 ± 16.83	655.75 ± 13.28	4.49 ± 0.25	0.033 ± 0.013
A	1047/890	890.33 ± 46.36 **	1031 ± 44.03 **, ##	1046.33 ± 38 **, ##	5.1 ± 0.12 **	0.014 ± 0.002 **
B	1133/920	920.6 ± 21.23 **	1014.2 ± 29.24 **, ##	1016.8 ± 28.4 **, ##	5.17 ± 0.1 **	0.015 ± 0.003 **
C	1203/999	999.2 ± 16.71 **	1098.6 ± 23.43 **	1109 ± 18.53 **	5.25 ± 0.07 **	0.014 ± 0.002 **

Note: The data were presented as the mean ± the SD (n = 3) compared to group CK, ** p < 0.01; and compared to group C, ^## p < 0.01.

). A total of 582 OTUs in all the rhizosphere samples were found in each group, and eight, 27, 64, 88, and 28 OTUs were exclusive to the rhizobacterial communities associated with group A, group B, group C, and group CK, respectively (Figure 5). The number of OTUs identified in the rhizosphere samples were as follows: group A, 837 to 921 (890.33 ± 46.36, n = 3); group B, 896 to 944 (920.6 ± 21.23, n = 5); group C, 981 to 1022 (999.2 ± 16.71, n = 5); and group CK, 559 to 585 (572.25 ± 12.26, n = 4).

The alpha diversity indices (Ace, Chao1, Shannon, and Simpson) were measured using the observed number of OTUs and showed significant changes (p < 0.01) in group A, group B, and group C compared to group CK (Table 1). For group A and group B, the Ace and Chao1 indices were significantly different compared to group C (p < 0.01). We speculated that the changes in the alpha diversity were related to the categories and dosages of the antibiotics selected in this study, and that the three antibiotics had different modes of action on the bacteria.

3.5. Impact of the Different Antibiotic Groups on the Rhizosphere Bacterial Community Structure

The community composition and relative abundance at different taxonomic levels (phylum and genus) are shown in Figure 6 and Figure 7, respectively.

At the phylum level, Actinobacteria, Proteobacteria, and Saccharibacteria were the dominant phyla. Ignavibacteriae was only found in group C (1.04%), and both Parcubacteria and Ignavibacteriae were not present in group CK. Although the most abundant bacterial groups in the antibiotic-adding groups were similar to group CK in the rhizosphere communities, the relative percentage exhibited a significant difference. At the phylum level, the Firmicutes were relatively less abundant and Saccharibacteria was more abundant in group CK compared to the three antibiotics groups. For Proteobacteria, the relative percentage in group A (53.20%) was significantly higher than in the other groups.

At the genus level, the top ten most common genera included Saccharibacteria_norank, Burkholderia–Paraburkholderia, Rhizomicrobium, Devosia, Bradyrhizobium, Chitinophagaceae_uncultured, Pseudolabrys, Mycobacterium, Gemmatimonadaceae_uncultured, and Rhodanobacter. Parcubacteria_norank was not present in group CK; however, BSV40_norank was found in group C. Most of the genera were in a lower abundance in the three antibiotic groups compared to group CK, and group C had a higher abundance than group A and group B. The results of ciprofloxacin on the bacterial community structure were not in agreement with the previous studies by Knecht et al. and Panda et al. [21,22].

The beta diversity was estimated using the unweighted UniFrac pairwise sample dissimilarity in the OTU abundance profiles among the samples. The unweighted UniFrac PCoA analysis revealed the differences in the community structures of group A, group B, group C, and group CK (Figure 8a). The rhizosphere soil samples of each group were tightly aggregated and separated on the first PCoA axis, which explained 58.10% of the variance. Consistent with the unweighted UniFrac PCoA results, the hierarchical tree-like structure also distinguished group CK from the three antibiotic groups (Figure 8b). The non-metric multidimensional scaling (NMDS) analysis revealed the distances among the samples in each group, as predicted (Figure 8c).

As determined by the LEfSe analysis, the rhizobacterial community composition differed among each group (Figure 9 and Figure 10). In group CK, Saccharibacteria and Saccharibacteria_g_norank were dominant at the phylum level and genus level, respectively, and the LDA scores were close to five. In the three antibiotics groups, Xanthomonadales and Gammaproteobacteria were dominant (LDA scores > 4) in group A; Firmicutes, Clostridia, and Clostridiales were the dominant phylum, class, and order, respectively, in group B; and Actinobacteria, Actinobacteria, and Micrococcales were the dominant phylum, class, and order in group C, respectively. From the above results, we concluded that the three antibiotics had a distinct effect on the different rhizobacterial community structures of Nicandra physalodes (Linn.) Gaertn.

The soil microbial community composition was dynamic and influenced by many factors, such as crop production practices, phospholipid fatty acid profiles, and substrate utilization patterns [23]. This study revealed the differences in the three antibiotic groups compared to group CK, and that the rhizosphere microbiome could indirectly reflect the antibiotic residues onto the plant performance and the rhizospheric organisms of Nicandra physalodes (Linn.) Gaertn., alerting people to the possible dangers of increasing serious antibacterial resistance. Ultimately, such knowledge could have a significant economic and environmental impact on the rational use of antibiotics in animal husbandry and veterinary science.