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Organic Houttuynia cordata Thunb harbors higher abundance and diversity of antibiotic resistance genes than non-organic origin, suggesting a potential food safe risk
Food Research International 120 (2019) 733–739
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Organic Houttuynia cordata Thunb harbors higher abundance and diversity of antibiotic resistance genes than non-organic origin, suggesting a potential food safe risk
T
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Wenliang Xianga, , Kekun Lua,b, Nandi Zhanga,b, Qianwen Lua,b, Qin Xua a b
School of Food and Bioengineering, Xihua University, Chengdu 610039, China Key Laboratory of Food Biotechnology of Sichuan, Chengdu 610039, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Houttuynia cordata Thunb Antibiotic resistance gene Bacterial community Co-occurrence between genes and bacteria
The organic agricultural products has been growing rapidly in recent years. However, a potential food safe risk, resulted by introduction more antibiotic resistant genes (ARGs) accompanied with animal manure using to organic farming, has long been overlooked. In current study, the bacterial community, 22 tetracycline, 3 aminoglycoside and 4 β-lactams ARGs were respectively investigated in the organic, chemical and wild Houttuynia cordata Thunb (HCT). A total of 9 tetracycline, 3 aminoglycoside and 2 β-lactam ARG subtypes were detected, and the organic HCT harbored more ARG subtypes. The absolute and relative abundance of total ARGs in organic HCT was strikingly higher than that in chemical and wild HCT. The Enterobacteriaceae, Aeromonadaceae, Pseudomonadceae, Moraxellaceae and Oxalobacteraceae were the dominant taxa in the chemical and wild HCT, but in the organic HCT, only Enterobacteriaceae posed 83.23% - 87.40% of bacterial community. Fourteen bacterial families might be the possible hosts of ARG subtypes in the HCT. Enterobacteriaceae was a possible host of most ARG subtypes, including tetA, tetB, tetC, tetE and aadA, and it was the main bacteria affecting the behavior of ARGs in the HCT. Additionally, the tetracycline ARG subtypes had more possible hosts. These results help to better understand the ARG potential food safe risk and develop effective measures to prevent the ARG dissemination in organic agricultural product.
1. Introduction The Houttuynia cordata Thunb (HCT), of family Saururaceae, is a well-known traditional edible and medicinal herb in Southeast Asia. Recently, it has already confirmed that HCT contains various pharmacological activities including diuretic, anti-bacterial, anti-viral, antitumor, anti-inflammatory, antioxidative, anti-diabetic, anti-allergic, and anti-mutagenic effects (Sekita et al., 2016). Thus, it has been recognized as an edible and medicinal resource with huge potential for development by the National Health Ministry of China and Japanese Pharmacopoeia guidebook. In China, HCT is used to preventing or remedying for treatment of various diseases or symptoms. However, with the increase of demand and irregular collection, wild HCT resource has greatly damaged. To meet the demand, HCT planting industry has quickly developed in the last few decades in China. Among these planting HCT, organic HCT delivers equally or more nutritious foods but with less or no pesticide residues and chemical fertilizers (Reganold & Wachter, 2016). Accordingly, there is a general perception that
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organic HCT is safer and healthier than non-organic origin. Commonly, concerns about the quality of organic food focus on the process-related quality and product criteria such as taste, nutrition and health, as well as organic specific indicators, but overlook the microbial safety, particularly regarding the presence of antibiotic resistant gene (ARG) (Fuentes, Morente, Abriouel, Pulido, & Gálvez, 2014). The discovery of ARG in organic food has already raised concern about the food safety of organic HCT. ARG is one of the most important emerging pollutants that threat to the food safety of organic product (Heuer, Schmitt, & Smalla, 2011). In organic farming, the animal manures are often recommended to replace chemical fertilizers. Unfortunately, these manures, especially those expose to the selection pressure of antibiotic residue, have been widely considered as a rich reservoir of ARGs (Zhu et al., 2013; Zhu, Chen, Chen, & Zhu, 2017). And accompanied with manure using,the ARGs are dragged into the organic farming, and then they will further spread in the organic agricultural ecosystem under the selection pressure of antibiotic residue in manures (Ji et al., 2012). The spread of ARGs is a
Corresponding author. E-mail address: [email protected] (W. Xiang).
https://doi.org/10.1016/j.foodres.2018.11.032 Received 19 July 2018; Received in revised form 14 October 2018; Accepted 16 November 2018 Available online 18 November 2018 0963-9969/ © 2018 Elsevier Ltd. All rights reserved.
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polyethylene bag on dry ice and analyzed immediately after arrival laboratory.
natural and ancient phenomenon that predates antibiotic using, but its high level prevalence in organic farming is generally considered as a modern phenomenon arising from animal manure using (Allen et al., 2010; Bush et al., 2011; D'Costa et al., 2011; Heuer et al., 2011; von Wintersdorff et al., 2016). Consumption of fresh plant product, particularly ready-to-eat vegetable, represents a route of human exposure to ARGs from the animal manures (Marti et al., 2013). Therefore, animal manures instead of chemical fertilizers are usually accompanied with a potential food safe threat resulting from ARG spread. Several studies have reported the presence of ARGs on the vegetable that grew in a manure-amended soil (Durso, Miller, & Wienhold, 2012; Wang, Qiao, Chen, Su, & Zhu, 2015). Raphael, Wong, and Riley (2011) reported that Gram-negative saprophytes isolated from retail organic than non-organic spinach carried extended-spectrum beta-lactamase (ESBL) genes, and the organically produced lettuce harbored higher abundance ARGs than conventionally produced (Zhu et al., 2017). But Ruimy et al. (2010) found that organic and non-organic vegetable contained equivalent counts of gram-negative antibiotic resistant bacteria, and the significant differences in antibiotic resistant pathogenic bacteria were not observed between various organic and non-organic leafy vegetables in Korea (Tango, Choi, Chung, & Oh, 2014). While it remains unknown whether the ARG in organic HCT is different from that in non-organic HCT or not. In current study, the ARGs in organic and non-organic HCT were evaluated and its potential host information was revealed through cooccurrence pattern between ARG subtypes and bacteria. To our knowledge, this is the first study to evaluate ARG risk factors with the microbial safety of traditional edible and medicinal HCT. The results facilitate better understanding of ARG potential food safe risks and providing further insights to devise effective preventive measures to improve the food safety of HCT.
2.2. DNA extraction Three replicates from each sampling site were equally mixed into test sample under sterile conditions. Then, about 5 g test samples were transferred into a 100 ml sterile centrifuge tube with 90 ml autoclaved 1 × phosphate buffered saline (PBS, pH 7.4) supplemented with 0.02% tween 20, and followed shaking at 200 rpm at 4 °C for 2 h. The washing solution was filtered with a sterilized nylon net and then centrifuged at 8000 rpm for 20 min at 4 °C. The resulting pellet was transferred into a 1.5 ml tube with 200 mg of zirconium beads (0.1 mm diameter). DNA extraction procedure was conducted as the soil DNA isolation kit protocol (Foregene, Chengdu, China). DNA concentration and quality were determined with a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and then it was stored at −80 °C until subsequent molecular analysis. 2.3. Bacterial community analysis using pyrosequencing The bacterial community structure was performed by 16S rRNA gene high-throughput sequencing. The V3 - V4 hyper-variable region was selected for PCR amplification with primers 341F (5′ - CCTAYGGGRBGCASCAG - 3′) and 806R (5′ - GGACTACNNGGGTATCTAAT - 3′). To pool all samples for a single pyrosequencing run, the reverse primer was tagged with unique barcodes. The thermal cycling conditions were as follows: 95 °C for 5 min, and 30 cycles of 95 °C for 60 s, 55 °C for 90 s and 72 °C for 60 s, then followed by 72 °C for 10 min. The negative control containing all components except for the template was also conducted. The amplified products were purified using gel-electrophoresis, and the desired fragments were sequenced on the Illumina Hiseq2500 platform (Majorbio, Shanghai, China). Then the high quality sequences, generated by Quantitative Insights Into Microbial Ecology (QIIME) software, were clustered into operation taxonomy units (OTUs) using UCLUST program at 97% similarity level. A representative sequence of each OTU was assigned to a taxonomic identity in the Ribosomal Database Project (http://rdp.cme.msu.edu/). Alpha diversity was described for each sample using observed species, Shannon indices and Chao1 indexes. Then the rarefaction curve was generated to compare the level of bacterial OTU diversity. The beta diversity of each HCT sample was also compared and visualized with principal coordinate analysis (PCoA) in QIIME based on Bray-Curtis distance.
2. Materials and methods 2.1. Sampling and pretreatment The HCT samples were collected from the different regions surrounding the Chengdu Plain in China in March 2016, including Penzhou mountainou region (30°59′24.72″N,103°57′28.10″E), Jintang hilly area (30°29′35.51″N,108°30′23.39″E), and Shifang flatland (31°7′36.51″N, 104°10′3.39″E). In these regions, the organic, chemical and wild HCTs were sampled by three replicates (Table 1), and the planting soil has been cultured for over 10 years. According to China national organic food standard (GB/T 19630.1–2011), chemical fertilizers, pesticides, growth regulating agents etc. are forbidden during the organic HCT production, and the bio-fertilizers derived from animal manures or manures mixed with crop residues must be fully composted before applied to the organic production systems. The sample for each replicate was respectively washed according to the ready-to-eat fresh vegetable standard (DB42/T 949–2014, China) and aseptically scissored into 2 cm segments by food handler, then it was kept in the sterile
2.4. PCR detection of ARGs Tetracycline, aminoglycoside and b-lactams were the largest consumption antibiotics in animal-farming in China. So the target chips of their ARG subtypes, including 22 tetracycline resistant genes, 3 aminoglycoside resistant genes and 4 β-lactams resistant genes, were respectively detected by PCR. The specific primers and amplification conditions were previously described by Ouoba, Lei, and Jensen (2008) for strA, strB and aadA; Tian, Zhang, Yu, and Yang (2016) for tetA; Jia et al. (2014) for tetB, tetC, tetD,tetE, tetG, tetL, tetO, tetQ, tetS, tetW and tetX; Jiang et al. (2013) for tetH, tetJ, tetK, tetY, tetZ and tet30; Zhang et al. (2016) for tetM, blaTEM and blaOXA-1; Aminov, GarriguesJeanjean, and Mackie (2001) for tetT and otrA; and Munoz, Benomar, Lerma, Galvez, and Abriouel (2014) for bla and blaZ (Table S1). Sterile water was used as the negative control in every run. After amplification, PCR products with the target genes were purified using universal DNA purification kits (Tiangen, Beijing, China). The purified segments were ligated into the pGEM-T plasmid vector (Promega, Madison, WI, USA) and then transformed into the chemically competent Escherichia coli JM109 cells (Promega, Madison, WI, USA). The positive clones were sequenced to further confirm whether PCR
Table 1 The HCT samples from three regions surrounding the Chengdu Flatland in China. Sample number
Product type
Growing area
CPH CSH CJH OPH OSH OJH WPH WSH WJH
Chemical planting HCT Chemical planting HCT Chemical planting HCT Organic planting HCT Organic planting HCT Organic planting HCT Wild collection HCT Wild collection HCT Wild collection HCT
Penzhou mountainous region Shifang flatland Jintang hilly area Penzhou mountainou region Shifang flatland Jintang hilly area Penzhou mountainou region Shifang flatland Jintang hilly area
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products were desired genes by BLAST search tool of Genbank database (http://www.ncbi.nlm.nih.gov/blast/), and those clones with ARG were then stored at −80 °C for subsequent qPCR assays. 2.5. Quantitative analysis of ARGs The qPCR was employed to quantify the ARG subtypes using aniCycler IQ5 Thermocycler (Bio-Rad, Hercules, CA, USA) and SYBR® Premix Ex TaqTM II (Takara, Dalian, China). The concentration and quality of plasmid DNA with target gene were quantified by the NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA). And the target gene copy number per microgram plasmid DNA was calculated as described by Zhang et al. (2016), where the lengths of the pGEM-T plasmid vector and target gene were already known. Then, 10-fold diluted plasmids, about from108 to 102 copies, were employed to generate the calibration curves. Based on the curves, the corresponding gene copies in HCT sample can be calculated. To avoid large fluctuations of ARG absolute abundance (ARG copy number per gram HCT), which caused by the differences in background bacterial abundance and DNA extraction efficiency, the ARG relative abundance (ARG copy number per 16S rRNA gene copy) was also used by normalizing their copy numbers to the bacterial 16S rRNA gene copy numbers for comparison in this study. The reaction was performed in 96 well plates with a final volume of 20 μL, containing 10 μL of SYBR Green Premix Ex Taq (Takara, Dalian, China), 0.4 μL primer (20 μM), 0.5 μL template DNA (20 ng/μL) and 9.1 μL ddH2O. The program was as follows: 95 °C for 4 min, followed by 35 cycles of 95 °C for 45 s, 60 °C for 45 s, 72 °C for 1 min, and the final stage of extending for 6 min at 72 °C (Wu, Qiao, Zhang, Cheng, & Zhu, 2010). The 16S rRNA qPCR was adapted from those reported by Tian et al. (2016). To ensure reproducibility, each reaction was run in triplicates. The PCR efficiency ranged from 80% to 110% with R2 values > 0.992 for all calibration curves. Data analysis was carried out with ICycler software. The specificity of qPCR products was assessed by melt curves from 60 to 95 °C. Fig. 1. Detected number (A) and abundance (B) of total ARGs in the chemical, organic and wild HCT samples from different regions.
2.6. Statistical analysis Statistical analysis was performed using Excel 2016 and SPSS 16.0. The correlation analysis was used to calculate the Pearson's bivariate correlation and p-values. One-way analysis of variance was used to assess the homogeneity of variance with significance level of 5% (p < .05). The redundancy analysis (RDA) in Canoco software V5.0 as a multivariate regression analysis was applied to show how the ARGs are related to bacterial community components. To visualize the correlations between ARG subtypes and bacterial taxa in the network interface, a correlation matrix was constructed by calculating all possible pairwise Spearman's rank correlations among the ARG subtypes. In addition, to reduce the chances of obtaining false-positive results, the Pvalues were adjusted with a multiple testing correction using the Benjamini-Hochberg method (Benjamini, Drai, Elmer, Kafkafi, & Golani, 2001). A correlation between two items was statistically robust if the Spearman's correlation coefficient (ρ) was > 0.8 and the P-value was < 0.01. Then, Cytoscape 3.3.0 was used to visualize the network graphs using Edge-weighted Spring Embedded Layout algorithms.
resistant bacteria can spread and persist in the phyllo- and rhizo-sphere of plant has raised concern about possible role of organic food in the spread of ARGs (Fuentes et al., 2014). In current investigation, all samples carried part of target ARGs, and there was no significant difference in the ARG number in the HCT from same production procedure but different regions. But the average number of ARGs in the organic HCT (ranging from 9 to 12) was significantly higher than that in nonorganic HCT (ranging from 2 to 3 in chemical and wild HCT) (Fig. 1A). The absolute and relative abundance of total ARGs in HCT samples were shown in Fig. 1B. In the organic HCT, they were respectively ranging from 5.76 to 6.30 log10 and −1.03 to −1.41 log10. Although the organic OJH harbored an absolute abundance of total ARGs that was 3 fold higher than that in the organic OPH and OSH, there was no significant difference of total ARG relative abundance among them. In three chemical HCTs, the absolute (from 2.98 to 3.21 log10) and relative abundance (from −3.47 to −3.1 log10) of total ARG had no significant difference. However, in the wild HCT from different production regions, there was marked difference in the absolute and relative abundance of total ARGs (Fig. 1B) (P > .05). The WSH from flatlands carried the absolute abundance of 4.85 log10 copies per gram sample and the relative abundance of −1.35 log10 copies per 16S rRNA gene copy, where they were respectively 2.16 log10 and 1.82 log10 fold higher than those in the WPH from mountainous region, and 1.85 log10 and 3.17 log10 fold higher than those in the WJH from hilly area. In general, the absolute and relative abundance of total ARGs in the organic HCTs were strikingly higher than those in chemical and wild HCT samples. And the high prevalence of total ARGs suggests an underlying
3. Results and discussion 3.1. Occurrence of total ARGs In recent years, human excessively irrational activities have made the natural ecosystem gradually aggravating. The wide application of antibiotics in agricultural production systems, especially in intensive animal farming, has already induced the evolution and spread of ARGs in soils (Ben, Wang, Pan, & Qiang, 2017; Cheng et al., 2016), and thus affects on the organic farming environment. The fact that antibiotic 735
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Fig. 2. The distribution and abundance of ARG subtypes in the chemical, organic and wild HCT samples. The different colored cells represent the log values of the abundance of ARG subtypes.
−1.47 log10 to −3.04 log10, −1.30 log10 to −3.03 log10, −1.07 log10 to −2.31 log10 copies per 16S rRNA gene (Fig. 2B), which was consistent with the reports that tetracycline resistant gene tetA, tetC and tetE were more often detected in wastewater or manure treatment plants (Xu et al., 2015; Zhu et al., 2017). Three ARG subtypes encoding aminoglycoside resistance, including nucleotidyl-transferase aadA gene, phosphotransferase strA and strB gene, were partly or completely detected in HCTs (Fig. 2). The aadA was found in all organic HCTs, and it had an absolute abundance of 4.57 log10 to 4.95 log10 copies per gram sample and a detection frequency of −2.61 log10 to −1.10 log10 copies per 16S rRNA gene. However, in the chemical and wild HCTs, only WPH carried aadA with an absolute abundance of 3.33 log10 copies per gram sample and with a detection frequency of −2.53 log10 copies per 16S rRNA gene (Fig. 2A and B). The strA and strB were both present in three organic samples, and except strB in OSH, their absolute and relative abundance were higher than those in chemical and wild HCT. Furthermore, the blaTEM and blaOXA-1 encoding beta lactamase were found in organic and chemical HCT samples, but not in wild HCT (Fig. 2A and B). The genes potentially conferring resistance to tetracycline, aminoglycoside and βlactam compounds were usually enriched most broadly in manure from swine feedlots (Zhu et al., 2013). These genes could shift to the farm ecosystem via discharge of animal wastes with simple treatment systems or even without any treatment, thus resulting in contamination of soil, water, and food resource (He et al., 2014; Liu et al., 2012). Bacterial traveling between soil and plant may be one of the reasons for the increased abundance of ARGs in the vegetable from organic production procedures. In addition, soil bacteria transmitted by aerosol could also serve as the inoculum source for plant microbes, and the increase of ARGs in vegetable could also be ascribed to the colonization of aerosol bacteria from soil during agricultural activities (Zhu et al., 2017).
food safe risk in the organic HCTs. 3.2. Diversity and abundance of ARG subtypes ARGs as emerging biological pollutants have drawn considerable attention due to their challenging clinical life-saving antibiotic therapies (Zhu et al., 2013). Fresh vegetables and fruits have been increasingly recognized as potential vehicles of ARG dissemination, and ARG occurrence and prevalence in them have already raised food safe concerns (Campos et al., 2013). In ARG subtype survey of HCTs, a total of 9 tetracycline, 3 aminoglycoside and 2 β-lactamase genes were detected (Fig. 2). The organic HCT harbored more ARG subtypes than chemical and wild HCT, suggesting that organic HCT had greater ARG diversity. The Simpson and Shannon indexes of ARG subtypes in the organic HCT were respectively 0.90–0.91 and 3.08–3.09, but those in the chemical and wild HCT were only 0.58–0.78 Simpson index and 0.96–1.56 Shannon index (Table S2). This result further demonstrated that the introduction of manure to organic farming could increase the potential safe risk of vegetable growing on manure-amended soil (Bai et al., 2015). The tetracycline resistant genes were divided into three categories, i) efflux pump genes, ii) ribosome protection genes and iii) inactivating enzyme genes. They have been already reported as the most frequently detected ARGs in the vegetables growing on manure-amended soil. (Raphael et al., 2011; Ruimy et al., 2010; Tango et al., 2014; Zhu et al., 2017). In current investigation, the tetracycline resistance genes including 6 efflux pump genes (tetA, tetB, tetC, tetD, tetE and tetG), 1 ribosomal protection protein gene (tetM) and 1 enzymatic modification gene (tetX) were detected in the organic HCT samples. While in chemical HCTs, only tetE and tetX genes were found, and there was none of tetracycline resistant genes in wild samples (Fig. 2).The tetA, tetC and tetE in the organic HCT had higher absolute abundance of 4.31 log10 to 5.32 log10, 4.15 log10 to 5.49 log10, 4.48 log10 to 6.28 log10 copies per gram sample than that in non-organic HCT (Fig. 2A). Meanwhile, they had also higher relative abundance (detection frequency) of
3.3. Characterization of bacterial community The soil may be a reservoir of the bacteria found in plants, and 736
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Fig. 4. Redundancy analysis (RDA) of the relationship between major microbial phyla (> 1% in any sample) and ARGs. Bacterial community data based on the different proportions of corresponding bacterial family in samples and ARG data based on the relative abundance of different antibiotics. A total of 96.90% variances can be explained by the selected variables.
intracellularly (Thomas & Nielsen, 2005). Thus, the change in bacterial community is prone to affect the proliferation and behavior of ARGs (Ben et al., 2017). Here RDA analysis confirmed that the ARG relative abundance was significantly correlated with bacterial community based on Bray-Curtis distance (r = 0.7833, P = .0002, permutations 9999). The 96.90% of the variance of ARG relative abundance could be explained by the selected variables with the first (77.12% of the total variance) and second axes (19.78% of the total variance) (Fig. 4). Enterobacteriaceae was observed to have a significant positive correlation with genes conferring resistance to tetracycline and β-lactams. Rizobacteria and Halomona were correlation with genes conferring resistance to aminoglycoside resistance (Fig. 4). These results suggested that Enterobacteriaceae was the main bacteria affecting the behavior of ARGs in the HCT. To further explore the possible hosts of ARGs, the non-random cooccurrence patterns were constructed by network analysis between the ARG subtypes and the bacterial taxa (at the family level). Previous studies confirmed that network analysis could provide new insights into ARGs and their possible hosts in complex environmental scenarios if the ARGs and the co-existing bacterial taxa possessed a strong and significantly positive correlation (Spearman's ρ > 0.8, P < .01) (Li et al., 2015; Zhang et al., 2016). In HCT, fourteen bacterial families were implicated in the possible hosts of ARG subtypes. Enterobacteriaceae was a possible host of most diverse ARG subtypes, including tetA, tetB, tetC, tetE and aadA, whereas Intraporangiaceae, Sphingobacteriaceae, Flavobacteriaceeae, Lactobacillaceae, Burkholderiaceae and Rhizobiaceae only had one resistance gene (Fig. 5). The tetracycline ARGs were observed to have more possible hosts, including Enterobacteriaceae, Moraxellaceae, Oxalobacteriaceae, Pseudomonadaceae, Comamonadaceae, Flavobacteriaceeae, Intraporangiaceae, Burkholderiaceae, Caulobacteraceae and Alteromonadaceae, while the aminoglycoside and β-lactams ARG subtypes only have one or two possible hosts (Fig. 5). In fact, network analysis was based on Spearman correlation and P value adjustment, the correlation between the two nodes (ARG subtype or bacterial taxa) merely depends on their abundance. Therefore to improve the robustness of network analysis in predicting the ARG host, further studies based on characterization of antibiotic resistant bacteria are indispensable in the future.
Fig. 3. Bacterial distribution heat map at family level (> 0.01% in any sample) in the chemical, organic and wild HCT samples. The different colored in the heat map means the different proportions of corresponding bacterial family in samples, respectively. 0.0001 in the scale means 0.01%.
bacterial traveling between soil and plant plays an important role in shaping bacterial community profile of plant (Bai et al., 2015). The organic production activities potentially provide a new disseminative way for bacteria from the livestock fecal in the agricultural environment and thus shift the characterization of bacterial community in natural plant (Zhu et al., 2017). The microbial community shift can affect the occurrence and abundance of ARGs (Cheng et al., 2016), but the information in vegetables is insufficient. The Illumina Hiseq2500 platform analysis showed that the bacterial community structure in the organic HCT was distinctly different from that in non-organic HCT (Adonis test, P < .05; Fig. 3 and Fig. S1). In the chemical and wild HCT, Enterobacteriaceae, Aeromonadaceae, Pseudomonadceae, Moraxellaceae and Oxalobacteraceae were the dominant taxa, accounting for approximately 85% of the whole bacterial community, while in the organic HCT, only Enterobacteriaceae possessed 83.23% - 87.40% of the whole bacterial community (Fig. 3). The rarefaction curves of OTUs at the sequencing depth of 24,780 showed that bacterial alpha-diversity decreased in organic HCT, which was further confirmed by the evaluation of Chao1 estimator, observed species and Shannon index (Fig. S2 and Table S2). The Enterobacteriaceae is a large family of Gram-negative bacteria, including most of fecal origin as well as some of non-fecal origin, and its presence in high numbers indicates a possible fecal contamination (Takahashi et al., 2017). Therefore, the application of animal manure may be one of the major reasons for increasing abundance of Enterobacteriaceae and decreasing the bacterial diversity in the organic HCT samples.
4. Conclusion This study revealed that organic HCT contained a higher abundance and diversity of ARGs, and the ARG increase was correlated with bacterial community shift. Enterobacteriaceae was a possible host of most diverse ARGs, including tetA, tetB, tetC tetE and aadA, and it was the main bacteria affecting the behavior of ARGs in the HCT. Recruitment
3.4. Correlation between ARGs and bacterial community ARGs can persist in extracellular gene elements such as plasmids and even naked DNA fragments, but their replications need to proceed 737
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Fig. 5. Network analysis revealing the co-occurrence patterns between ARGs subtypes and bacterial taxa (family level). The nodes were colored according to ARG subtypes and bacterial taxa. The connection between ARG subtypes and bacterial taxa represents a strong (Spearman's correlation coefficient ρ > 0.8) and significant (P-value < 0.01) correlation.
of ARGs-harboring bacteria from manure-fertilized soil may be one of the main reasons for the higher abundance and diversity of ARGs in organic HCT. It should be noted that the higher vegetable-associated ARGs could pose a potential risk to human health, especially for readyto-eat fresh vegetable. This study implies that more investigations are warranted in preventing the ARG potential safe risks of consuming ready-to-eat fresh organic vegetable.
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Acknowledgment This work was supported by the National Natural Science Foundation of China, China (31571935), the Applied Basic Research Program of Sichuan, China (2018JY0045) and the General Program of Chengdu, China (2016-NY02-00064-NC). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodres.2018.11.032. References Allen, H. K., Donato, J., Wang, H. H., Cloud-Hansen, K. A., Davies, J., & Handelsman, J. (2010). Call of the wild: Antibiotic resistance genes in natural environments. Nature Reviews Microbiology, 8(4), 251–259. Aminov, R. I., Garrigues-Jeanjean, N., & Mackie, R. I. (2001). Molecular ecology of tetracycline resistance: Development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins. Applied and Environmental Microbiology, 67(1), 22–32. Bai, Y., Muller, D. B., Srinivas, G., Garrido-Oter, R., Potthoff, E., Rott, M., ... SchulzeLefert, P. (2015). Functional overlap of the Arabidopsis leaf and root microbiota. Nature, 528(7582), 364–369. Ben, W. W., Wang, J., Pan, X., & Qiang, Z. M. (2017). Dissemination of antibiotic resistance genes and their potential removal by on-farm treatment processes in nine swine feedlots in Shandong Province, China. Chemosphere, 167, 262–268. Benjamini, Y., Drai, D., Elmer, G., Kafkafi, N., & Golani, I. (2001). Controlling the false discovery rate in behavior genetics research. Behavioural Brain Research, 125(1–2), 279–284. Bush, K., Courvalin, P., Dantas, G., Davies, J., Eisenstein, B., Huovinen, P., ... Zgurskaya, H. I. (2011). Tackling antibiotic resistance. Nature Reviews Microbiology, 9(12), 894–896. Campos, J., Mourao, J., Pestana, N., Peixe, L., Novais, C., & Antunes, P. (2013).
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digestion from mesophilic to thermophilic. Water Research, 98, 261–269. Wang, F. H., Qiao, M., Chen, Z., Su, J. Q., & Zhu, Y. G. (2015). Antibiotic resistance genes in manure-amended soil and vegetables at harvest. Journal of Hazardous Materials, 299, 215–221. von Wintersdorff, C. J. H., Penders, J., van Niekerk, J. M., Mills, N. D., Majumder, S., van Alphen, L. B., ... Wolffs, P. F. G. (2016). Dissemination of antimicrobial resistance in microbial ecosystems through horizontal gene transfer. Frontiers in Microbiology, 7(173). Wu, N., Qiao, M., Zhang, B., Cheng, W. D., & Zhu, Y. G. (2010). Abundance and diversity of tetracycline resistance genes in soils adjacent to representative swine feedlots in China. Environmental Science and Technology, 44(18), 6933–6939. Xu, J., Xu, Y., Wang, H. M., Guo, C. S., Qiu, H. Y., He, Y., ... Meng, W. (2015). Occurrence of antibiotics and antibiotic resistance genes in a sewage treatment plant and its effluent-receiving river. Chemosphere, 119, 1379–1385. Zhang, J. Y., Chen, M. X., Sui, Q. W., Wang, R., Tong, J., & Wei, Y. S. (2016). Fate of antibiotic resistance genes and its drivers during anaerobic co-digestion of food waste and sewage sludge based on microwave pretreatment. Bioresource Technology, 217, 28–36. Zhu, B., Chen, Q., Chen, S., & Zhu, Y. G. (2017). Does organically produced lettuce harbor higher abundance of antibiotic resistance genes than conventionally produced? Environment International, 98, 152–159. Zhu, Y. G., Johnson, T. A., Su, J. Q., Qiao, M., Guo, G. X., Stedtfeld, R. D., ... Tiedje, J. M. (2013). Diverse and abundant antibiotic resistance genes in Chinese swine farms. Proceedings of the National Academy of Sciences of the United States of America, 110(9), 3435–3440.
bacteria and bifidobacteria of African and European origin to antimicrobials: Determination and transferability of the resistance genes to other bacteria. International Journal of Food Microbiology, 121(2), 217–224. Raphael, E., Wong, L. K., & Riley, L. W. (2011). Extended-spectrum beta-lactamase gene sequences in gram-negative saprophytes on retail organic and nonorganic spinach. Applied and Environmental Microbiology, 77(5), 1601–1607. Reganold, J. P., & Wachter, J. M. (2016). Organic agriculture in the twenty-first century. Nature Plants, 2(2), 15221. Ruimy, R., Brisabois, A., Bernede, C., Skurnik, D., Barnat, S., Arlet, G., ... Andremont, A. (2010). Organic and conventional fruits and vegetables contain equivalent counts of gram-negative bacteria expressing resistance to antibacterial agents. Environmental Microbiology, 12(3), 608–615. Sekita, Y., Murakami, K., Yumoto, H., Mizuguchi, H., Amoh, T., Ogino, S., ... Kashiwada, Y. (2016). Anti-bacterial and anti-inflammatory effects of ethanol extract from Houttuynia cordata poultice. Bioscience, Biotechnology, and Biochemistry, 80(6), 1205–1213. Takahashi, H., Saito, R., Miya, S., Tanaka, Y., Miyamura, N., Kuda, T., & Kimura, B. (2017). Development of quantitative real-time PCR for detection and enumeration of Enterobacteriaceae. International Journal of Food Microbiology, 246, 92–97. Tango, C. N., Choi, N. J., Chung, M. S., & Oh, D. H. (2014). Bacteriological quality of vegetables from organic and conventional production in different areas of Korea. Journal of Food Protection, 77(8), 1411–1417. Thomas, C. M., & Nielsen, K. M. (2005). Mechanisms of, and barriers to, horizontal gene transfer between bacteria. Nature Reviews Microbiology, 3(9), 711–721. Tian, Z., Zhang, Y., Yu, B., & Yang, M. (2016). Changes of resistome, mobilome and potential hosts of antibiotic resistance genes during the transformation of anaerobic
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Organic Houttuynia cordata Thunb harbors higher abundance and diversity of antibiotic resistance genes than non-organic origin, suggesting a potential food safe risk
T
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Wenliang Xianga, , Kekun Lua,b, Nandi Zhanga,b, Qianwen Lua,b, Qin Xua a b
School of Food and Bioengineering, Xihua University, Chengdu 610039, China Key Laboratory of Food Biotechnology of Sichuan, Chengdu 610039, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Houttuynia cordata Thunb Antibiotic resistance gene Bacterial community Co-occurrence between genes and bacteria
The organic agricultural products has been growing rapidly in recent years. However, a potential food safe risk, resulted by introduction more antibiotic resistant genes (ARGs) accompanied with animal manure using to organic farming, has long been overlooked. In current study, the bacterial community, 22 tetracycline, 3 aminoglycoside and 4 β-lactams ARGs were respectively investigated in the organic, chemical and wild Houttuynia cordata Thunb (HCT). A total of 9 tetracycline, 3 aminoglycoside and 2 β-lactam ARG subtypes were detected, and the organic HCT harbored more ARG subtypes. The absolute and relative abundance of total ARGs in organic HCT was strikingly higher than that in chemical and wild HCT. The Enterobacteriaceae, Aeromonadaceae, Pseudomonadceae, Moraxellaceae and Oxalobacteraceae were the dominant taxa in the chemical and wild HCT, but in the organic HCT, only Enterobacteriaceae posed 83.23% - 87.40% of bacterial community. Fourteen bacterial families might be the possible hosts of ARG subtypes in the HCT. Enterobacteriaceae was a possible host of most ARG subtypes, including tetA, tetB, tetC, tetE and aadA, and it was the main bacteria affecting the behavior of ARGs in the HCT. Additionally, the tetracycline ARG subtypes had more possible hosts. These results help to better understand the ARG potential food safe risk and develop effective measures to prevent the ARG dissemination in organic agricultural product.
1. Introduction The Houttuynia cordata Thunb (HCT), of family Saururaceae, is a well-known traditional edible and medicinal herb in Southeast Asia. Recently, it has already confirmed that HCT contains various pharmacological activities including diuretic, anti-bacterial, anti-viral, antitumor, anti-inflammatory, antioxidative, anti-diabetic, anti-allergic, and anti-mutagenic effects (Sekita et al., 2016). Thus, it has been recognized as an edible and medicinal resource with huge potential for development by the National Health Ministry of China and Japanese Pharmacopoeia guidebook. In China, HCT is used to preventing or remedying for treatment of various diseases or symptoms. However, with the increase of demand and irregular collection, wild HCT resource has greatly damaged. To meet the demand, HCT planting industry has quickly developed in the last few decades in China. Among these planting HCT, organic HCT delivers equally or more nutritious foods but with less or no pesticide residues and chemical fertilizers (Reganold & Wachter, 2016). Accordingly, there is a general perception that
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organic HCT is safer and healthier than non-organic origin. Commonly, concerns about the quality of organic food focus on the process-related quality and product criteria such as taste, nutrition and health, as well as organic specific indicators, but overlook the microbial safety, particularly regarding the presence of antibiotic resistant gene (ARG) (Fuentes, Morente, Abriouel, Pulido, & Gálvez, 2014). The discovery of ARG in organic food has already raised concern about the food safety of organic HCT. ARG is one of the most important emerging pollutants that threat to the food safety of organic product (Heuer, Schmitt, & Smalla, 2011). In organic farming, the animal manures are often recommended to replace chemical fertilizers. Unfortunately, these manures, especially those expose to the selection pressure of antibiotic residue, have been widely considered as a rich reservoir of ARGs (Zhu et al., 2013; Zhu, Chen, Chen, & Zhu, 2017). And accompanied with manure using,the ARGs are dragged into the organic farming, and then they will further spread in the organic agricultural ecosystem under the selection pressure of antibiotic residue in manures (Ji et al., 2012). The spread of ARGs is a
Corresponding author. E-mail address: [email protected] (W. Xiang).
https://doi.org/10.1016/j.foodres.2018.11.032 Received 19 July 2018; Received in revised form 14 October 2018; Accepted 16 November 2018 Available online 18 November 2018 0963-9969/ © 2018 Elsevier Ltd. All rights reserved.
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polyethylene bag on dry ice and analyzed immediately after arrival laboratory.
natural and ancient phenomenon that predates antibiotic using, but its high level prevalence in organic farming is generally considered as a modern phenomenon arising from animal manure using (Allen et al., 2010; Bush et al., 2011; D'Costa et al., 2011; Heuer et al., 2011; von Wintersdorff et al., 2016). Consumption of fresh plant product, particularly ready-to-eat vegetable, represents a route of human exposure to ARGs from the animal manures (Marti et al., 2013). Therefore, animal manures instead of chemical fertilizers are usually accompanied with a potential food safe threat resulting from ARG spread. Several studies have reported the presence of ARGs on the vegetable that grew in a manure-amended soil (Durso, Miller, & Wienhold, 2012; Wang, Qiao, Chen, Su, & Zhu, 2015). Raphael, Wong, and Riley (2011) reported that Gram-negative saprophytes isolated from retail organic than non-organic spinach carried extended-spectrum beta-lactamase (ESBL) genes, and the organically produced lettuce harbored higher abundance ARGs than conventionally produced (Zhu et al., 2017). But Ruimy et al. (2010) found that organic and non-organic vegetable contained equivalent counts of gram-negative antibiotic resistant bacteria, and the significant differences in antibiotic resistant pathogenic bacteria were not observed between various organic and non-organic leafy vegetables in Korea (Tango, Choi, Chung, & Oh, 2014). While it remains unknown whether the ARG in organic HCT is different from that in non-organic HCT or not. In current study, the ARGs in organic and non-organic HCT were evaluated and its potential host information was revealed through cooccurrence pattern between ARG subtypes and bacteria. To our knowledge, this is the first study to evaluate ARG risk factors with the microbial safety of traditional edible and medicinal HCT. The results facilitate better understanding of ARG potential food safe risks and providing further insights to devise effective preventive measures to improve the food safety of HCT.
2.2. DNA extraction Three replicates from each sampling site were equally mixed into test sample under sterile conditions. Then, about 5 g test samples were transferred into a 100 ml sterile centrifuge tube with 90 ml autoclaved 1 × phosphate buffered saline (PBS, pH 7.4) supplemented with 0.02% tween 20, and followed shaking at 200 rpm at 4 °C for 2 h. The washing solution was filtered with a sterilized nylon net and then centrifuged at 8000 rpm for 20 min at 4 °C. The resulting pellet was transferred into a 1.5 ml tube with 200 mg of zirconium beads (0.1 mm diameter). DNA extraction procedure was conducted as the soil DNA isolation kit protocol (Foregene, Chengdu, China). DNA concentration and quality were determined with a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and then it was stored at −80 °C until subsequent molecular analysis. 2.3. Bacterial community analysis using pyrosequencing The bacterial community structure was performed by 16S rRNA gene high-throughput sequencing. The V3 - V4 hyper-variable region was selected for PCR amplification with primers 341F (5′ - CCTAYGGGRBGCASCAG - 3′) and 806R (5′ - GGACTACNNGGGTATCTAAT - 3′). To pool all samples for a single pyrosequencing run, the reverse primer was tagged with unique barcodes. The thermal cycling conditions were as follows: 95 °C for 5 min, and 30 cycles of 95 °C for 60 s, 55 °C for 90 s and 72 °C for 60 s, then followed by 72 °C for 10 min. The negative control containing all components except for the template was also conducted. The amplified products were purified using gel-electrophoresis, and the desired fragments were sequenced on the Illumina Hiseq2500 platform (Majorbio, Shanghai, China). Then the high quality sequences, generated by Quantitative Insights Into Microbial Ecology (QIIME) software, were clustered into operation taxonomy units (OTUs) using UCLUST program at 97% similarity level. A representative sequence of each OTU was assigned to a taxonomic identity in the Ribosomal Database Project (http://rdp.cme.msu.edu/). Alpha diversity was described for each sample using observed species, Shannon indices and Chao1 indexes. Then the rarefaction curve was generated to compare the level of bacterial OTU diversity. The beta diversity of each HCT sample was also compared and visualized with principal coordinate analysis (PCoA) in QIIME based on Bray-Curtis distance.
2. Materials and methods 2.1. Sampling and pretreatment The HCT samples were collected from the different regions surrounding the Chengdu Plain in China in March 2016, including Penzhou mountainou region (30°59′24.72″N,103°57′28.10″E), Jintang hilly area (30°29′35.51″N,108°30′23.39″E), and Shifang flatland (31°7′36.51″N, 104°10′3.39″E). In these regions, the organic, chemical and wild HCTs were sampled by three replicates (Table 1), and the planting soil has been cultured for over 10 years. According to China national organic food standard (GB/T 19630.1–2011), chemical fertilizers, pesticides, growth regulating agents etc. are forbidden during the organic HCT production, and the bio-fertilizers derived from animal manures or manures mixed with crop residues must be fully composted before applied to the organic production systems. The sample for each replicate was respectively washed according to the ready-to-eat fresh vegetable standard (DB42/T 949–2014, China) and aseptically scissored into 2 cm segments by food handler, then it was kept in the sterile
2.4. PCR detection of ARGs Tetracycline, aminoglycoside and b-lactams were the largest consumption antibiotics in animal-farming in China. So the target chips of their ARG subtypes, including 22 tetracycline resistant genes, 3 aminoglycoside resistant genes and 4 β-lactams resistant genes, were respectively detected by PCR. The specific primers and amplification conditions were previously described by Ouoba, Lei, and Jensen (2008) for strA, strB and aadA; Tian, Zhang, Yu, and Yang (2016) for tetA; Jia et al. (2014) for tetB, tetC, tetD,tetE, tetG, tetL, tetO, tetQ, tetS, tetW and tetX; Jiang et al. (2013) for tetH, tetJ, tetK, tetY, tetZ and tet30; Zhang et al. (2016) for tetM, blaTEM and blaOXA-1; Aminov, GarriguesJeanjean, and Mackie (2001) for tetT and otrA; and Munoz, Benomar, Lerma, Galvez, and Abriouel (2014) for bla and blaZ (Table S1). Sterile water was used as the negative control in every run. After amplification, PCR products with the target genes were purified using universal DNA purification kits (Tiangen, Beijing, China). The purified segments were ligated into the pGEM-T plasmid vector (Promega, Madison, WI, USA) and then transformed into the chemically competent Escherichia coli JM109 cells (Promega, Madison, WI, USA). The positive clones were sequenced to further confirm whether PCR
Table 1 The HCT samples from three regions surrounding the Chengdu Flatland in China. Sample number
Product type
Growing area
CPH CSH CJH OPH OSH OJH WPH WSH WJH
Chemical planting HCT Chemical planting HCT Chemical planting HCT Organic planting HCT Organic planting HCT Organic planting HCT Wild collection HCT Wild collection HCT Wild collection HCT
Penzhou mountainous region Shifang flatland Jintang hilly area Penzhou mountainou region Shifang flatland Jintang hilly area Penzhou mountainou region Shifang flatland Jintang hilly area
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products were desired genes by BLAST search tool of Genbank database (http://www.ncbi.nlm.nih.gov/blast/), and those clones with ARG were then stored at −80 °C for subsequent qPCR assays. 2.5. Quantitative analysis of ARGs The qPCR was employed to quantify the ARG subtypes using aniCycler IQ5 Thermocycler (Bio-Rad, Hercules, CA, USA) and SYBR® Premix Ex TaqTM II (Takara, Dalian, China). The concentration and quality of plasmid DNA with target gene were quantified by the NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA). And the target gene copy number per microgram plasmid DNA was calculated as described by Zhang et al. (2016), where the lengths of the pGEM-T plasmid vector and target gene were already known. Then, 10-fold diluted plasmids, about from108 to 102 copies, were employed to generate the calibration curves. Based on the curves, the corresponding gene copies in HCT sample can be calculated. To avoid large fluctuations of ARG absolute abundance (ARG copy number per gram HCT), which caused by the differences in background bacterial abundance and DNA extraction efficiency, the ARG relative abundance (ARG copy number per 16S rRNA gene copy) was also used by normalizing their copy numbers to the bacterial 16S rRNA gene copy numbers for comparison in this study. The reaction was performed in 96 well plates with a final volume of 20 μL, containing 10 μL of SYBR Green Premix Ex Taq (Takara, Dalian, China), 0.4 μL primer (20 μM), 0.5 μL template DNA (20 ng/μL) and 9.1 μL ddH2O. The program was as follows: 95 °C for 4 min, followed by 35 cycles of 95 °C for 45 s, 60 °C for 45 s, 72 °C for 1 min, and the final stage of extending for 6 min at 72 °C (Wu, Qiao, Zhang, Cheng, & Zhu, 2010). The 16S rRNA qPCR was adapted from those reported by Tian et al. (2016). To ensure reproducibility, each reaction was run in triplicates. The PCR efficiency ranged from 80% to 110% with R2 values > 0.992 for all calibration curves. Data analysis was carried out with ICycler software. The specificity of qPCR products was assessed by melt curves from 60 to 95 °C. Fig. 1. Detected number (A) and abundance (B) of total ARGs in the chemical, organic and wild HCT samples from different regions.
2.6. Statistical analysis Statistical analysis was performed using Excel 2016 and SPSS 16.0. The correlation analysis was used to calculate the Pearson's bivariate correlation and p-values. One-way analysis of variance was used to assess the homogeneity of variance with significance level of 5% (p < .05). The redundancy analysis (RDA) in Canoco software V5.0 as a multivariate regression analysis was applied to show how the ARGs are related to bacterial community components. To visualize the correlations between ARG subtypes and bacterial taxa in the network interface, a correlation matrix was constructed by calculating all possible pairwise Spearman's rank correlations among the ARG subtypes. In addition, to reduce the chances of obtaining false-positive results, the Pvalues were adjusted with a multiple testing correction using the Benjamini-Hochberg method (Benjamini, Drai, Elmer, Kafkafi, & Golani, 2001). A correlation between two items was statistically robust if the Spearman's correlation coefficient (ρ) was > 0.8 and the P-value was < 0.01. Then, Cytoscape 3.3.0 was used to visualize the network graphs using Edge-weighted Spring Embedded Layout algorithms.
resistant bacteria can spread and persist in the phyllo- and rhizo-sphere of plant has raised concern about possible role of organic food in the spread of ARGs (Fuentes et al., 2014). In current investigation, all samples carried part of target ARGs, and there was no significant difference in the ARG number in the HCT from same production procedure but different regions. But the average number of ARGs in the organic HCT (ranging from 9 to 12) was significantly higher than that in nonorganic HCT (ranging from 2 to 3 in chemical and wild HCT) (Fig. 1A). The absolute and relative abundance of total ARGs in HCT samples were shown in Fig. 1B. In the organic HCT, they were respectively ranging from 5.76 to 6.30 log10 and −1.03 to −1.41 log10. Although the organic OJH harbored an absolute abundance of total ARGs that was 3 fold higher than that in the organic OPH and OSH, there was no significant difference of total ARG relative abundance among them. In three chemical HCTs, the absolute (from 2.98 to 3.21 log10) and relative abundance (from −3.47 to −3.1 log10) of total ARG had no significant difference. However, in the wild HCT from different production regions, there was marked difference in the absolute and relative abundance of total ARGs (Fig. 1B) (P > .05). The WSH from flatlands carried the absolute abundance of 4.85 log10 copies per gram sample and the relative abundance of −1.35 log10 copies per 16S rRNA gene copy, where they were respectively 2.16 log10 and 1.82 log10 fold higher than those in the WPH from mountainous region, and 1.85 log10 and 3.17 log10 fold higher than those in the WJH from hilly area. In general, the absolute and relative abundance of total ARGs in the organic HCTs were strikingly higher than those in chemical and wild HCT samples. And the high prevalence of total ARGs suggests an underlying
3. Results and discussion 3.1. Occurrence of total ARGs In recent years, human excessively irrational activities have made the natural ecosystem gradually aggravating. The wide application of antibiotics in agricultural production systems, especially in intensive animal farming, has already induced the evolution and spread of ARGs in soils (Ben, Wang, Pan, & Qiang, 2017; Cheng et al., 2016), and thus affects on the organic farming environment. The fact that antibiotic 735
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Fig. 2. The distribution and abundance of ARG subtypes in the chemical, organic and wild HCT samples. The different colored cells represent the log values of the abundance of ARG subtypes.
−1.47 log10 to −3.04 log10, −1.30 log10 to −3.03 log10, −1.07 log10 to −2.31 log10 copies per 16S rRNA gene (Fig. 2B), which was consistent with the reports that tetracycline resistant gene tetA, tetC and tetE were more often detected in wastewater or manure treatment plants (Xu et al., 2015; Zhu et al., 2017). Three ARG subtypes encoding aminoglycoside resistance, including nucleotidyl-transferase aadA gene, phosphotransferase strA and strB gene, were partly or completely detected in HCTs (Fig. 2). The aadA was found in all organic HCTs, and it had an absolute abundance of 4.57 log10 to 4.95 log10 copies per gram sample and a detection frequency of −2.61 log10 to −1.10 log10 copies per 16S rRNA gene. However, in the chemical and wild HCTs, only WPH carried aadA with an absolute abundance of 3.33 log10 copies per gram sample and with a detection frequency of −2.53 log10 copies per 16S rRNA gene (Fig. 2A and B). The strA and strB were both present in three organic samples, and except strB in OSH, their absolute and relative abundance were higher than those in chemical and wild HCT. Furthermore, the blaTEM and blaOXA-1 encoding beta lactamase were found in organic and chemical HCT samples, but not in wild HCT (Fig. 2A and B). The genes potentially conferring resistance to tetracycline, aminoglycoside and βlactam compounds were usually enriched most broadly in manure from swine feedlots (Zhu et al., 2013). These genes could shift to the farm ecosystem via discharge of animal wastes with simple treatment systems or even without any treatment, thus resulting in contamination of soil, water, and food resource (He et al., 2014; Liu et al., 2012). Bacterial traveling between soil and plant may be one of the reasons for the increased abundance of ARGs in the vegetable from organic production procedures. In addition, soil bacteria transmitted by aerosol could also serve as the inoculum source for plant microbes, and the increase of ARGs in vegetable could also be ascribed to the colonization of aerosol bacteria from soil during agricultural activities (Zhu et al., 2017).
food safe risk in the organic HCTs. 3.2. Diversity and abundance of ARG subtypes ARGs as emerging biological pollutants have drawn considerable attention due to their challenging clinical life-saving antibiotic therapies (Zhu et al., 2013). Fresh vegetables and fruits have been increasingly recognized as potential vehicles of ARG dissemination, and ARG occurrence and prevalence in them have already raised food safe concerns (Campos et al., 2013). In ARG subtype survey of HCTs, a total of 9 tetracycline, 3 aminoglycoside and 2 β-lactamase genes were detected (Fig. 2). The organic HCT harbored more ARG subtypes than chemical and wild HCT, suggesting that organic HCT had greater ARG diversity. The Simpson and Shannon indexes of ARG subtypes in the organic HCT were respectively 0.90–0.91 and 3.08–3.09, but those in the chemical and wild HCT were only 0.58–0.78 Simpson index and 0.96–1.56 Shannon index (Table S2). This result further demonstrated that the introduction of manure to organic farming could increase the potential safe risk of vegetable growing on manure-amended soil (Bai et al., 2015). The tetracycline resistant genes were divided into three categories, i) efflux pump genes, ii) ribosome protection genes and iii) inactivating enzyme genes. They have been already reported as the most frequently detected ARGs in the vegetables growing on manure-amended soil. (Raphael et al., 2011; Ruimy et al., 2010; Tango et al., 2014; Zhu et al., 2017). In current investigation, the tetracycline resistance genes including 6 efflux pump genes (tetA, tetB, tetC, tetD, tetE and tetG), 1 ribosomal protection protein gene (tetM) and 1 enzymatic modification gene (tetX) were detected in the organic HCT samples. While in chemical HCTs, only tetE and tetX genes were found, and there was none of tetracycline resistant genes in wild samples (Fig. 2).The tetA, tetC and tetE in the organic HCT had higher absolute abundance of 4.31 log10 to 5.32 log10, 4.15 log10 to 5.49 log10, 4.48 log10 to 6.28 log10 copies per gram sample than that in non-organic HCT (Fig. 2A). Meanwhile, they had also higher relative abundance (detection frequency) of
3.3. Characterization of bacterial community The soil may be a reservoir of the bacteria found in plants, and 736
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Fig. 4. Redundancy analysis (RDA) of the relationship between major microbial phyla (> 1% in any sample) and ARGs. Bacterial community data based on the different proportions of corresponding bacterial family in samples and ARG data based on the relative abundance of different antibiotics. A total of 96.90% variances can be explained by the selected variables.
intracellularly (Thomas & Nielsen, 2005). Thus, the change in bacterial community is prone to affect the proliferation and behavior of ARGs (Ben et al., 2017). Here RDA analysis confirmed that the ARG relative abundance was significantly correlated with bacterial community based on Bray-Curtis distance (r = 0.7833, P = .0002, permutations 9999). The 96.90% of the variance of ARG relative abundance could be explained by the selected variables with the first (77.12% of the total variance) and second axes (19.78% of the total variance) (Fig. 4). Enterobacteriaceae was observed to have a significant positive correlation with genes conferring resistance to tetracycline and β-lactams. Rizobacteria and Halomona were correlation with genes conferring resistance to aminoglycoside resistance (Fig. 4). These results suggested that Enterobacteriaceae was the main bacteria affecting the behavior of ARGs in the HCT. To further explore the possible hosts of ARGs, the non-random cooccurrence patterns were constructed by network analysis between the ARG subtypes and the bacterial taxa (at the family level). Previous studies confirmed that network analysis could provide new insights into ARGs and their possible hosts in complex environmental scenarios if the ARGs and the co-existing bacterial taxa possessed a strong and significantly positive correlation (Spearman's ρ > 0.8, P < .01) (Li et al., 2015; Zhang et al., 2016). In HCT, fourteen bacterial families were implicated in the possible hosts of ARG subtypes. Enterobacteriaceae was a possible host of most diverse ARG subtypes, including tetA, tetB, tetC, tetE and aadA, whereas Intraporangiaceae, Sphingobacteriaceae, Flavobacteriaceeae, Lactobacillaceae, Burkholderiaceae and Rhizobiaceae only had one resistance gene (Fig. 5). The tetracycline ARGs were observed to have more possible hosts, including Enterobacteriaceae, Moraxellaceae, Oxalobacteriaceae, Pseudomonadaceae, Comamonadaceae, Flavobacteriaceeae, Intraporangiaceae, Burkholderiaceae, Caulobacteraceae and Alteromonadaceae, while the aminoglycoside and β-lactams ARG subtypes only have one or two possible hosts (Fig. 5). In fact, network analysis was based on Spearman correlation and P value adjustment, the correlation between the two nodes (ARG subtype or bacterial taxa) merely depends on their abundance. Therefore to improve the robustness of network analysis in predicting the ARG host, further studies based on characterization of antibiotic resistant bacteria are indispensable in the future.
Fig. 3. Bacterial distribution heat map at family level (> 0.01% in any sample) in the chemical, organic and wild HCT samples. The different colored in the heat map means the different proportions of corresponding bacterial family in samples, respectively. 0.0001 in the scale means 0.01%.
bacterial traveling between soil and plant plays an important role in shaping bacterial community profile of plant (Bai et al., 2015). The organic production activities potentially provide a new disseminative way for bacteria from the livestock fecal in the agricultural environment and thus shift the characterization of bacterial community in natural plant (Zhu et al., 2017). The microbial community shift can affect the occurrence and abundance of ARGs (Cheng et al., 2016), but the information in vegetables is insufficient. The Illumina Hiseq2500 platform analysis showed that the bacterial community structure in the organic HCT was distinctly different from that in non-organic HCT (Adonis test, P < .05; Fig. 3 and Fig. S1). In the chemical and wild HCT, Enterobacteriaceae, Aeromonadaceae, Pseudomonadceae, Moraxellaceae and Oxalobacteraceae were the dominant taxa, accounting for approximately 85% of the whole bacterial community, while in the organic HCT, only Enterobacteriaceae possessed 83.23% - 87.40% of the whole bacterial community (Fig. 3). The rarefaction curves of OTUs at the sequencing depth of 24,780 showed that bacterial alpha-diversity decreased in organic HCT, which was further confirmed by the evaluation of Chao1 estimator, observed species and Shannon index (Fig. S2 and Table S2). The Enterobacteriaceae is a large family of Gram-negative bacteria, including most of fecal origin as well as some of non-fecal origin, and its presence in high numbers indicates a possible fecal contamination (Takahashi et al., 2017). Therefore, the application of animal manure may be one of the major reasons for increasing abundance of Enterobacteriaceae and decreasing the bacterial diversity in the organic HCT samples.
4. Conclusion This study revealed that organic HCT contained a higher abundance and diversity of ARGs, and the ARG increase was correlated with bacterial community shift. Enterobacteriaceae was a possible host of most diverse ARGs, including tetA, tetB, tetC tetE and aadA, and it was the main bacteria affecting the behavior of ARGs in the HCT. Recruitment
3.4. Correlation between ARGs and bacterial community ARGs can persist in extracellular gene elements such as plasmids and even naked DNA fragments, but their replications need to proceed 737
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Fig. 5. Network analysis revealing the co-occurrence patterns between ARGs subtypes and bacterial taxa (family level). The nodes were colored according to ARG subtypes and bacterial taxa. The connection between ARG subtypes and bacterial taxa represents a strong (Spearman's correlation coefficient ρ > 0.8) and significant (P-value < 0.01) correlation.
of ARGs-harboring bacteria from manure-fertilized soil may be one of the main reasons for the higher abundance and diversity of ARGs in organic HCT. It should be noted that the higher vegetable-associated ARGs could pose a potential risk to human health, especially for readyto-eat fresh vegetable. This study implies that more investigations are warranted in preventing the ARG potential safe risks of consuming ready-to-eat fresh organic vegetable.
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