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Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Microbial response to repeated treatments of manure containing sulfadiazine and chlortetracycline in soil a
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Hua Fang , Yu L. Han , Yuan M. Yin , Xiang X. Jin , Shao Y. Wang , Fei F. Tang , Lin Cai & a
Yun L. Yu a
Institute of Pesticide and Environmental Toxicology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China b
Environmental Biotechnology Laboratory, Department of Civil Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China Published online: 05 Jun 2014.
Click for updates To cite this article: Hua Fang, Yu L. Han, Yuan M. Yin, Xiang X. Jin, Shao Y. Wang, Fei F. Tang, Lin Cai & Yun L. Yu (2014) Microbial response to repeated treatments of manure containing sulfadiazine and chlortetracycline in soil, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 49:8, 609-615, DOI: 10.1080/03601234.2014.911592 To link to this article: http://dx.doi.org/10.1080/03601234.2014.911592
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Journal of Environmental Science and Health, Part B (2014) 49, 609–615 Copyright © Taylor & Francis Group, LLC ISSN: 0360-1234 (Print); 1532-4109 (Online) DOI: 10.1080/03601234.2014.911592
Microbial response to repeated treatments of manure containing sulfadiazine and chlortetracycline in soil HUA FANG1, YU L. HAN1, YUAN M. YIN1, XIANG X. JIN1, SHAO Y. WANG1, FEI F. TANG1, LIN CAI2 and YUN L. YU1 1
Institute of Pesticide and Environmental Toxicology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China Environmental Biotechnology Laboratory, Department of Civil Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China
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Substantive addition of antibiotic-contaminated manure to agricultural soil may lead to “persistent” residues of antibiotics and may affect soil health. Therefore, this study examines the effects of repeated manure treatments containing sulfadiazine (SDZ) and chlortetracycline (CTC) residues, both individually and combined, on the functional diversity and structure of soil microbial communities in the soils under laboratory conditions. The average well color development (AWCD), Simpson diversity index (1/D, dominant populations), Shannon-Wiener diversity index (H0 , richness), and McIntosh diversity index (U, evenness) in the antibiotics-treated soils decreased in the first 60-day treatment and then gradually recovered or even exceeded the initial level in the unamended soils with increasing treatment frequency. A total of 11 specific bands in temperature gradient gel electrophoresis (TGGE) profiles were observed and sequence analyzed for five repeated treatments, and most of them belonged to the phyla Firmicutes, Actinobacteria, and Proteobacteria. These results indicate that repeated treatments of manure containing SDZ and CTC residues can alter soil microbial community structure, although they have a temporary suppression effect on soil microbial functional diversity. Keywords: Sulfadiazine, chlortetracycline, repeated treatment, functional diversity, community structure.
Introduction Since the early 1990s, antibiotics in China have been widely used in livestock industries as therapeutic agents for infectious diseases and growth promoters.[1] It is estimated that approximately 30–90% of antibiotics fed to livestock could be excreted as parent compound or metabolites by animal feces or urine. Therefore, antibiotics and their metabolites entered soil environments through the practice of spreading livestock wastes as base fertilizer onto agricultural fields several times a year to enhance crop yields.[2–4] Due to the rapid dissipation of antibiotics in agricultural soil, their residual levels are not high, ranging from mg kg–1 to mg kg–1.[5] However, repeated use of antibiotic-contaminated manure could lead to “quasi-persistent” antibiotic residues and antibiotic resistance, as
Address correspondence to Yun L. Yu, Institute of Pesticide and Environmental Toxicology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China; E-mail: [email protected] Received December 17, 2013.
well as to altered soil microbial community structure and function, ultimately posing an ecological and environmental risk to soil quality and human health.[2,6,7] Therefore, there is an increasing interest in investigating the effects of repeated antibiotic-contaminated manure treatments on the functional diversity and community structure of soil microorganisms. Sulfadiazine (SDZ) is a sulfonamide antibiotic that is widely applied as a veterinary antibiotic to prevent and treat diarrhea and other infectious disease.[8] Chlortetracycline (CTC) is a tetracycline antibiotic that is widely used as a feed additive to promote growth in calves, pigs, and poultry.[8] The residues of SDZ or CTC are frequently detected in environmental compartments and substrates, such as manure, biosolid, soil, food plants, surface water, and groundwater.[9] The effects of single treatment of SDZ or CTC on the functional and structural diversity of soil microorganisms have been well investigated.[10,11] Sulfonamide antibiotics were reported to affect overall microbial activities and the bacterial community structure,[12] and shifts from bacteria to fungi were observed.[13] The effects of repeated treatments of SDZ- or CTC-contaminated manure might be different from single treatment.
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Therefore, it is necessary to investigate the effects of repeated treatments of SDZ and CTC, both individually and combined, on soil microbial communities in manured soil. In this study, pig manure containing SDZ and CTC, both individually and combined, was repeatedly added to the soil. The objectives of this study were (1) to examine different substrate utilization patterns by soil microorganisms; (2) to measure responses of soil microbial functional diversity; and (3) to reveal the changes in soil microbial community structure. The results obtained will help assess the environmental safety associated with successive fertilization with antibiotics-contaminated manure in agricultural soil.
triplicate and incubated at 25 § 1 C in the dark. Five successive additions of SDZ and CTC were performed every 60 days. At the end of each treatment, 15.0 g of soil samples was collected from each plastic frame to determine soil microbial functional diversity. Sixty days after each treatment and 120 days after the final treatment, 10.0 g of soil samples was collected to determine bacterial community structure.
Materials and methods
DNA extraction and PCR-TGGE
Chemicals
The soil used in this study was collected from a mulberry field located at the Huajiachi Campus of Zhejiang University, Hangzhou, China. The soil had not been contaminated by SDZ and CTC. The soil samples were sieved (2 mm) to remove stones and debris. The soil texture was classified as a silt loam by its physicochemical properties (sand, 21.5%; silt, 71.1%; clay, 7.4%; organic matter content, 3.05%; water holding capacity, 39.4%; cationic exchange capacity, 10.6 cmol kg–1; total nitrogen, 0.14%; and pH 6.8). The pig manure was collected from a piggery located at the Huajiachi Campus of Zhejiang University, and manure chemical properties were determined as follows: soluble organic matter, 385.4 g kg–1; total nitrogen, 53.4 g kg–1; P2O5, 27.6 g kg–1; and pH 5.9.
Total soil DNA was extracted from each sample with a FastDNA Spin Kit according to the manufacturer’s protocol (Qbiogene, Carlsbad, CA, USA) and was then used as a template to amplify the 16S rDNA V3 region using the primer set GC341F and 518R with a TPersonal Thermalcycler (Whatman Biometra, Gottingen, Germany). PCR was conducted according to the method of Wang et al.[15] PCR products were analyzed by 1.0% agarose gel electrophoresis with TAE buffer containing 1.0 mg mL–1 ethidium bromide. The temperature gradient gel electrophoresis (TGGE) Mini system (Biometra, Gottingen, Germany) was used for sequence-specific separation of PCR products. Polyacrylamide gels were composed of 8% acrylamide/bis (37.5:1), 8 mol L–1 urea, 20% formamide and 2% glycerol. The electrophoresis was performed at a constant voltage of 200 V for 3 h in 1 £ TAE buffer (40 mmol L–1 Tris-Acetate, pH 8.0). A temperature gradient of 42 C to 50 C in parallel with the direction of electrophoresis was used. After the electrophoresis, gels were stained with AgNO3 as described by the manufacturer (Biometra). The stained gels were immediately photographed using a digital camera DSC-F717 (Sony, Tokyo, Japan).
Soil treatment and sampling
Phylogenetic analysis
Pig manure (80 g) was first suspended in sterile water, and SDZ and CTC in methanol solutions were successively added separately and together. Next, the pig manure was thoroughly mixed with the soil (2.0 kg dry weight equivalent) to obtain a normal agricultural manure amount of 4% (w/w) and a final antibiotic concentration of 20.0 mg kg–1 of dry soil. Finally, the soil mixtures were sieved (2-mm mesh) and transferred to a 25-L plastic frame covered with aluminum foil. Soil samples amended with the same volume water were used as untreated control. Soil water content was adjusted to 60% of the maximum water holding capacity and maintained by periodic distilled water additions. All treatments were carried out in
The specific bands on TGGE gels were cut out with sterile razor blades and eluted in 30 mL of sterile double-deionized H2O overnight at 4 C. Approximately 3 mL of elution was used as a PCR template under the conditions described above. The purified PCR products were ligated into a pMD19-T vector (TaKaRa, Dalian, China) according to the manufacturer’s recommendations, transformed into E. coli DH5a and then sequenced (Invitrogen, Shanghai, China). The obtained 16S rDNA sequences were submitted to the NCBI GenBank to identify the nearest genera or species according to the BLAST program. A phylogenetic tree of these sequences was constructed by MEGA5.05, with Thermocrinis ruber DSM 12173 used as the outgroup.[16]
Technical grade SDZ (purity 98.5%) and CTC (purity 98.5%) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Soils and manure
Carbon substrate utilization Carbon substrate utilization by soil microorganisms during repeated antibiotic treatments was determined according to the method described by Fang et al.[14]
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Statistical analysis The absorbance values at 72 h were used to calculate the Simpson diversity index (1/D), Shannon-Wiener index (H0 ), and McIntosh index (U) according to Eqs. (1)–(3): DD
X ðniðni ¡ 1ÞÞ
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ðN ðN ¡ 1ÞÞ X 0 H D¡ piðlnpiÞ rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi X ffi UD ni2
(1) (2) (3)
where ni is the absorbance value, N is the total absorbance value of all wells, pi is the proportional absorbance value of the well over the total absorbance value of all wells, and the Simpson index is the reciprocal (1/D). The 1/D, H0 , and U indices are used to assess the dominant population, richness, and evenness of soil microorganisms, respectively. Diversity index differences between different treatments were analyzed by one-way ANOVA using SPSS 11.5 software (SPSS Inc., Chicago, IL, USA).
Results Substrate utilization patterns of soil microorganisms The average well color development (AWCD) in the BIOLOG ECO plate, reflecting the oxidative capacity of soil microbial communities, usually serves as an indicator of overall microbial activities. As shown in Fig. 1, in the first 60-day treatment, the antibiotics treatment AWCD values were lower than the individual manure treatment values. In the second 60-day treatment, there was no significant difference observed in the AWCD values among all treatments. However, in the third, fourth, and fifth 60day treatments, the antibiotics treatment AWCD values were higher than those of the individual manure treatment. ANOVA showed that both the CTC treatment and SDZ C CTC treatment significantly (P 0.05) enhanced the AWCD values of soil microbial communities after the third treatment. However, no significant difference in the AWCD values was found between the individual manure treatment and SDZ treatment. Response of soil microbial functional diversity to repeated treatments The effects of repeated SDZ and CTC treatments on soil microbial functional diversity are summarized in Table 1. In the first 60-day treatment, the 1/D, H0 , and U indices decreased by 27.0%, 12.5%, and 28.2% in the SDZ treatment, 30.3%, 14.9%, and 40.8% in the CTC treatment, and 41.5%, 18.0%, and 29.6% in the SDZ C CTC treatment, respectively, compared with those in the
Fig. 1. AWCD variations throughout the incubation period for soil microbial communities during repeated treatments of SDZ and/or CTC.
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Table 1 Effects of repeated treatments of SDZ and/or CTC treatments on the functional diversity of soil microorganisms.
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Treatment frequency 1st 1st 1st 1st 1st 2nd 2nd 2nd 2nd 2nd 3rd 3rd 3rd 3rd 3rd 4th 4th 4th 4th 4th 5th 5th 5th 5th 5th
Time (days)
Treatment
Simpson index (1/D)
Shannon-Wiener index (H0 )
McIntosh index (U)
60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60
CK Manure SDZ CTC SDZCCTC CK Manure SDZ CTC SDZCCTC CK Manure SDZ CTC SDZCCTC CK Manure SDZ CTC SDZCCTC CK Manure SDZ CTC SDZCCTC
8.16 § 0.72 c 15.27 § 1.31 a 11.14 § 0.97 b 10.65 § 0.46 b 8.93 § 0.33 c 4.98 § 0.45 c 8.71 § 0.85 a 7.64 § 0.53 b 7.80 § 0.74 b 8.04 § 0.87 ab 6.62 § 0.45 e 7.52 § 0.74 d 9.43 § 0.45 c 10.41 § 1.02 b 12.43 § 1.83 a 9.26 § 0.43 b 9.34 § 0.83 b 10.13 § 0.98 a 10.51 § 0.52 a 10.76 § 0.99 a 5.99 § 0.75 e 6.07 § 0.45 d 8.04 § 0.46 c 9.55 § 0.74 b 11.07 § 1.02 a
2.23 § 0.08 b 2.89 § 0.08 a 2.53 § 0.06 ab 2.46 § 0.07 ab 2.37 § 0.06 ab 1.90 § 0.07 b 2.47 § 0.08 a 1.92 § 0.08 b 2.36 § 0.05 a 2.45 § 0.08 a 2.18 § 0.05 b 2.25 § 0.08 ab 2.41 § 0.06 a 2.62 § 0.07 a 2.68 § 0.04 a 2.39 § 0.08 a 2.41 § 0.07 a 2.48 § 0.07 a 2.61 § 0.05 a 2.61 § 0.08 a 2.08 § 0.08 b 2.02 § 0.08 b 2.42 § 0.05 a 2.50 § 0.07 a 2.65 § 0.09 a
1.10 § 0.09 b 2.13 § 0.24 a 1.53 § 0.07 b 1.26 § 0.08 b 1.50 § 0.10 b 0.89 § 0.07 b 1.94 § 0.08 a 1.08 § 0.04 b 1.27 § 0.07 b 0.97 § 0.09 b 1.63 § 0.12 c 1.37 § 0.05 c 1.45 § 0.08 c 2.96 § 0.15 a 2.14 § 0.10 b 1.42 § 0.09 b 1.59 § 0.12 b 1.84 § 0.10 b 1.78 § 0.11 b 2.51 § 0.16 a 2.21 § 0.10 a 1.31 § 0.09 b 2.02 § 0.12 a 2.74 § 0.15 a 2.58 § 0.20 a
Data followed by a different letter in the same column are significantly different (P 0.05).
individual manure treatment. In the second 60-day treatment, the 1/D and H0 indices in the antibiotic-treated soils showed a recovery, but the U index was still significantly smaller compared with individual manure treatment levels. The 1/D index in the SDZ, CTC, SDZCCTC treatments significantly (P 0.05) increased by 25.4%, 38.4%, and 65.3% in the third treatment, 8.5%, 12.5%, and 15.2% in the fourth treatment, and 32.5%, 57.3%, and 82.4% in the fifth treatment, respectively, and the H0 and U indices also slightly exceeded those in the individual manure treatment.
As shown in Fig. 2, the PC1 component (49.0%) in the PCA profile showed more important power than the PC2 component (13.7%). In the first 60-day treatment, a dispersed pattern was observed, and antibiotic treatment scores in the PC1 axis were lower than those in the individual manure treatment. This dispersed pattern was retained in the second and third 60-day treatments, and the scores of the antibiotic treatments in the PC1 axis were similar to those of the individual manure treatment. In the fourth and fifth 60-day treatments, a grouped pattern was gradually formed.
Response of soil microbial community structure to repeated treatments
Fig. 2. PCA analysis of soil microbial communities during the five repeated treatments of SDZ and/or CTC.
The TGGE profiles of 16S rDNA V3 regions derived from all treatments are shown in Fig. 3. Throughout this study, no obvious changes in the TGGE banding patterns were observed in the unamended control. During the five repeated treatments, more bands were detected in the manured soils than in the control soils. Furthermore, some specific bands were found between the individual manure soil and the antibiotics-treated soil. As shown in Fig. 3, specific bands 1–11 were selected, cut, and sequenced to identify soil microbial populations (the sequences of the 11 bands are summarized in Table S1). The
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Fig. 3. TGGE analysis of 16S rDNA fragments derived from the five repeated treatments of SDZ and/or CTC. Lanes 1–5: CK, Manure, SDZ, CTC, and SDZCCTC.
phylogeneticrelationshipbetweenthese11sequencesandreference sequences obtained from NCBI GenBank is presented in Fig. 4, and their phylogenetic affiliations are summarized in Table 2. In the first 60-day treatment, the dominant band 11 belonged to the phylum Actinobacteria and showed a high similarity (100%) to Rhodococcus equi strain DSM 20307. In
Fig. 4. Neighbor-joining tree showing the phylogenetic affiliations of 16S rDNA V3 sequences obtained from bands excised from TGGE gels during five repeated treatments of SDZ and/or CTC. The tree was rooted with the partial 16S rDNA sequence of Thermocrinis ruber DSM 12173 as an outgroup. Values at nodes represent the percentage of 1,000 bootstrap replicates. The scale bar indicates an estimated change of 5%.
the second 60-day treatment, the intensity of band 9 was much higher in the antibiotics-treated soils than in the control soils, and it belonged to the phylum Firmicutes and showed a high similarity (100%) to Clostridium disporicum strain DS1. Band10intheSDZtreatmentbelongedtothephylumActinobacteria and showed 97% similarity to Microlunatus aurantiacus strain YIM 45721, but it was not found in the CTC and SDZCCTC treatments. In the third 60-day treatment, the intensity of band 2 in the antibiotic treatments was higher than in the other treatments, which belonged to the phylum Proteobacteriaandshowed98%similaritytoSulfurovumlithotrophicum stain 42BKT. In the fourth 60-day treatment, band 3 was observed only in the antibiotic treatments affiliated with the phylum Firmicutes and showed 99% similarity to Dehalogenimonas lykanthroporepellens strain BL-DC-9. Inthefifth60-daytreatment,dominantbands5and8,belonging to the phyla Firmicutes and Proteobacteria, specifically formed in the antibiotic treatments. The intensity of band 7, belonging to Tenericutes, was much higher in the fifth treatment than in previous treatments, but it was not found in the CTCtreatment. In the 120 days after the final treatment, the intensities of bands 3, 4, and 9 (all of them belonging to the phylum Firmicutes) became lower than those in the five previous treatments. However, bands 5, 7, and 8 were still retained with high intensities in the TGGE profiles.
Discussion The decreased AWCD values from antibiotics-treated soils during the first treatment may be due to the suppression of bacteria sensitive to SDZ or CTC. In agreement with this result, Liu et al.[11] reported that six types of antibiotics (CTC, tetracycline, tylosin, sulfamethoxazole,
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Table 2 Phylogenetic affiliations of 16S rDNA V3 sequences excised from TGGE gels during five repeated treatments of SDZ and/or CTC. Band no.
Fragment length (bp)
Band 1 Band 2 Band 3 Band 4 Band 5 Band 6 Band 7 Band 8 Band 9 Band 10 Band 11
172 170 170 195 170 171 170 172 170 175 175
Closest relative in GenBank Sphaerobacter thermophilus strain DSM 20745 Sulfurovum lithotrophicum strain 42BKT Dehalogenimonas lykanthroporepellens strain BL-DC-9 Bacillus niabensis strain 4T19 Clostridium quinii strain DSM 6736 Clostridium cellulovorans strain 743B Mycoplasma glycophilum strain 486 Anaerobiospirillum succiniciproducens strain S411 Clostridium disporicum strain DS1 Microlunatus aurantiacus strain YIM 45721 Rhodococcus equi strain DSM 20307
sulfamethazine, and trimethoprim) could suppress soil microbial activities. Girardi et al.[17] revealed that ciprofloxacin strongly inhibited microbial activities. Liu et al.[18] reported that 100 mg kg–1 of sulfamethoxazole had a significant inhibitory effect on AWCD values after a 7-day treatment. In this study, however, the significant increase of AWCD values in CTC and SDZCCTC treatments in the third treatment indicated that CTC might have a stimulatory effect on soil microbial activities, possibly due to the formation and proliferation of tolerant or resistant bacteria. A similar finding was also reported by Liu et al.[19] that oxytetracycline could increase the AWCD values in a 7-week greenhouse pot experiment with BIOLOG ECO microplates. In this study, the AWCD values from the manured soils were much higher than those from the unamended control soils. This phenomenon is not surprising because manure application could increase the soil nutrition and lead to a microbial biomass increase.[20,21] During the first treatment, the 1/D, H0 , and U indices decreased significantly (P 0.05) in the antibiotic treatments, which indicated that the dominant populations, richness, and evenness of soil microorganisms were suppressed by SDZ or CTC. Similar to this finding, Kong et al.[22] reported that the H0 and U indices decreased significantly (P 0.005) with increasing concentrations of oxytetracycline. Liu et al.[18] reported that the H0 index could be suppressed by 100 mg kg–1 of sulfamethoxazole after a 7-day treatment. In contrast, Muller et al.[23] revealed no H0 index differences in tylosin-treated soils. PCA analysis showed a trend from dispersed patterns to grouped patterns, indicating an initial suppression of sensitive bacteria by antibiotics, but then, repeated treatments stimulated growth of resistant bacteria and improved microbial activities. As shown in the TGGE profiles, the total number of bands in the manure treatment was considerably higher than in the manure-unamended treatment. A similar phenomenon was reported by Hammesfahr et al.[10,20] who showed that manure had a stimulating effect on soil microbial communities such as PLFAtot and could increase band
Similarity
Putative phylum
99% 98% 99% 100% 98% 87% 94% 88% 100% 97% 100%
Actinobacteria Proteobacteria Firmicutes Firmicutes Firmicutes Firmicutes Tenericutes Proteobacteria Firmicutes Actinobacteria Actinobacteria
intensity. In this study, repeated treatments of SDZ and/ or CTC altered soil microbial community structure. Hammesfahr et al.[10] reported that a single application of SDZ in combination with manure had a prolonged effect on the microbial community structure, resulting in altered community structure. In this study, band 10 occurred in the SDZ treatment but was not found in the CTC and SDZCCTC treatments, suggesting that CTC has an inhibitory effect on such bacteria. The high intensity of bands 2, 3, 5, and 8 in the antibiotic treatments indicated that SDZ or CTC caused the formation and proliferation of these resistant bacteria. Band 7 occurred in the SDZ and SDZCCTC treatments but was not found in the CTC treatment, indicating that SDZ may have a stimulatory effect on these microbial species. Some studies reported that SDZ treatment induced the formation of additional bands, representing related resistant bacteria.[12,24] Hammesfahr et al.[20] reported that microbial structure was more sensitive to SDZ contamination than functional processes, and the amendment of pig liquid manure and SDZ altered microbial community structure. Furthermore, the effect of SDZ on microbial community structure increased with incremental liquid manure amendment. The findings in this study indicate that the functional diversity of the soil microbial community was temporarily inhibited during the first treatment but gradually recovered with further treatments. Repeated treatments of SDZ and/ or CTC altered soil microbial community structure. Therefore, repeated treatments of antibiotics may have a persistent impact on soil health. Further studies need to be conducted to reveal the diversity, abundance, and expression level of resistant genes in the antibiotic-contaminated soils.
Funding This work was supported by the National High Technology R&D Program of China (No. 2012AA06A204), the
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Microbial response to repeated treatments of manure National Nature Science Foundation of China (No. 21377112, 20907040), and the Public Science and Technology Research Funds Projects of the Ministry of Environmental Protection of China (No. 201109018).
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Supplemental Material Supplemental data for this article can be accessed on the publisher’s website.
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the microbial community structure in agricultural soils. Soil Biol. Biochem. 2008, 40(7), 1583–1591. Liu, F.; Ying, G.G.; Tao, R.; Zhao, J.L.; Yang, J.F.; Zhao, L.F. Effects of six selected antibiotics on plant growth and soil microbial and enzymatic activities. Environ. Pollut. 2009, 157(5), 1636–1642. Zielezny, Y.; Groeneweg, J.; Vereecken, H.; Tappe, W. Impact of sulfadiazine and chlorotetracycline on soil bacterial community structure and respiratory activity. Soil Biol. Biochem. 2006, 38(8), 2372–2380. Thiele-Bruhn, S.; Beck, I.C. Effects of sulfonamide and tetracycline antibiotics on soil microbial activity and microbial biomass. Chemosphere. 2005, 59(4), 457–465. Fang, H.; Tang, F.F.; Zhou, W.; Cao, Z.Y.; Wang, D.D.; Liu, K. L.; Wu, X.W.; Yu, Y.L. Persistence of repeated triadimefon application and its impact on soil microbial functional diversity. J. Environ. Sci. Heal. B 2012, 47(2), 104–110. Wang, X.G.; Song, M.; Wang, Y.Q.; Gao, C.M.; Zhang, Q.; Chu, X.Q.; Fang, H.; Yu, Y.L. Response of soil bacterial community to repeated applications of carbendazim. Ecotox. Environ. Safe. 2012, 75, 33–39. Tamura, K.; Peterson, D.; Peterson, N.; Stecher, G.; Nei, M.; Kumar, S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.Mol.Biol.Evol.2011,28(10), 2731–2739. Girardi, C.; Greve, J.; Lamshoft, M.; Fetzer, I.; Miltner, A.; Schaffer A, Kastner M. Biodegradation of ciprofloxacin in water and soil and its effects on the microbial communities. J. Hazard. Mater. 2011, 198, 22–30. Liu, F.; Wu, J.S.; Ying, G.G.; Luo, Z.X.; Feng, H. Changes in functional diversity of soil microbial community with addition of antibiotics sulfamethoxazole and chlortetracycline. Appl. Microbiol. Biotechnol. 2012, 95(6), 1615–1623. Liu, W.; Pan, N.; Chen, W.; Jiao, W.; Wang, M. Effect of veterinary oxytetracycline on functional diversity of soil microbial community. Plant Soil Environ. 2012, 58(7), 295–301. Hammesfahr, U.; Bierl, R.; Thiele-Bruhn, S. Combined effects of the antibiotic sulfadiazine and liquid manure on the soil microbialcommunity structure and functions. J. Plant Nutr. Soil Sci. 2011, 174(4), 614–623. Kotzerke, A.; Hammesfahr, U.; Kleineidam, K.; Lamshoft, M.; Thiele-Bruhn, S.; Schloter, M.; Wilke, B.M. Influence of difloxacin-contaminated manure on microbial community structure and function in soils. Biol. Fertil. Soils 2011, 47(2), 177–186. Kong, W.D.; Zhu, Y.G.; Fu, B.J.; Marschner, P.; He, J.Z. The veterinary antibiotic oxytetracycline and Cu influence functional diversity of the soil microbial community. Environ. Pollut. 2006, 143(1), 129–137. Muller, A.K.; Westergaard, K.; Christensen, S.; Sorensen, S.J. The diversity and function of soil microbial communities exposed to different disturbances. Microb. Ecol. 2002, 44, 49–58. Heuer, H.; Smalla, K. Manure and sulfadiazine synergistically increased bacterial antibiotic resistance in soil over at least two months. Environ. Microbiol. 2007, 9(3), 657–666.
Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Microbial response to repeated treatments of manure containing sulfadiazine and chlortetracycline in soil a
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Hua Fang , Yu L. Han , Yuan M. Yin , Xiang X. Jin , Shao Y. Wang , Fei F. Tang , Lin Cai & a
Yun L. Yu a
Institute of Pesticide and Environmental Toxicology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China b
Environmental Biotechnology Laboratory, Department of Civil Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China Published online: 05 Jun 2014.
Click for updates To cite this article: Hua Fang, Yu L. Han, Yuan M. Yin, Xiang X. Jin, Shao Y. Wang, Fei F. Tang, Lin Cai & Yun L. Yu (2014) Microbial response to repeated treatments of manure containing sulfadiazine and chlortetracycline in soil, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 49:8, 609-615, DOI: 10.1080/03601234.2014.911592 To link to this article: http://dx.doi.org/10.1080/03601234.2014.911592
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Journal of Environmental Science and Health, Part B (2014) 49, 609–615 Copyright © Taylor & Francis Group, LLC ISSN: 0360-1234 (Print); 1532-4109 (Online) DOI: 10.1080/03601234.2014.911592
Microbial response to repeated treatments of manure containing sulfadiazine and chlortetracycline in soil HUA FANG1, YU L. HAN1, YUAN M. YIN1, XIANG X. JIN1, SHAO Y. WANG1, FEI F. TANG1, LIN CAI2 and YUN L. YU1 1
Institute of Pesticide and Environmental Toxicology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China Environmental Biotechnology Laboratory, Department of Civil Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China
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Substantive addition of antibiotic-contaminated manure to agricultural soil may lead to “persistent” residues of antibiotics and may affect soil health. Therefore, this study examines the effects of repeated manure treatments containing sulfadiazine (SDZ) and chlortetracycline (CTC) residues, both individually and combined, on the functional diversity and structure of soil microbial communities in the soils under laboratory conditions. The average well color development (AWCD), Simpson diversity index (1/D, dominant populations), Shannon-Wiener diversity index (H0 , richness), and McIntosh diversity index (U, evenness) in the antibiotics-treated soils decreased in the first 60-day treatment and then gradually recovered or even exceeded the initial level in the unamended soils with increasing treatment frequency. A total of 11 specific bands in temperature gradient gel electrophoresis (TGGE) profiles were observed and sequence analyzed for five repeated treatments, and most of them belonged to the phyla Firmicutes, Actinobacteria, and Proteobacteria. These results indicate that repeated treatments of manure containing SDZ and CTC residues can alter soil microbial community structure, although they have a temporary suppression effect on soil microbial functional diversity. Keywords: Sulfadiazine, chlortetracycline, repeated treatment, functional diversity, community structure.
Introduction Since the early 1990s, antibiotics in China have been widely used in livestock industries as therapeutic agents for infectious diseases and growth promoters.[1] It is estimated that approximately 30–90% of antibiotics fed to livestock could be excreted as parent compound or metabolites by animal feces or urine. Therefore, antibiotics and their metabolites entered soil environments through the practice of spreading livestock wastes as base fertilizer onto agricultural fields several times a year to enhance crop yields.[2–4] Due to the rapid dissipation of antibiotics in agricultural soil, their residual levels are not high, ranging from mg kg–1 to mg kg–1.[5] However, repeated use of antibiotic-contaminated manure could lead to “quasi-persistent” antibiotic residues and antibiotic resistance, as
Address correspondence to Yun L. Yu, Institute of Pesticide and Environmental Toxicology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China; E-mail: [email protected] Received December 17, 2013.
well as to altered soil microbial community structure and function, ultimately posing an ecological and environmental risk to soil quality and human health.[2,6,7] Therefore, there is an increasing interest in investigating the effects of repeated antibiotic-contaminated manure treatments on the functional diversity and community structure of soil microorganisms. Sulfadiazine (SDZ) is a sulfonamide antibiotic that is widely applied as a veterinary antibiotic to prevent and treat diarrhea and other infectious disease.[8] Chlortetracycline (CTC) is a tetracycline antibiotic that is widely used as a feed additive to promote growth in calves, pigs, and poultry.[8] The residues of SDZ or CTC are frequently detected in environmental compartments and substrates, such as manure, biosolid, soil, food plants, surface water, and groundwater.[9] The effects of single treatment of SDZ or CTC on the functional and structural diversity of soil microorganisms have been well investigated.[10,11] Sulfonamide antibiotics were reported to affect overall microbial activities and the bacterial community structure,[12] and shifts from bacteria to fungi were observed.[13] The effects of repeated treatments of SDZ- or CTC-contaminated manure might be different from single treatment.
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Therefore, it is necessary to investigate the effects of repeated treatments of SDZ and CTC, both individually and combined, on soil microbial communities in manured soil. In this study, pig manure containing SDZ and CTC, both individually and combined, was repeatedly added to the soil. The objectives of this study were (1) to examine different substrate utilization patterns by soil microorganisms; (2) to measure responses of soil microbial functional diversity; and (3) to reveal the changes in soil microbial community structure. The results obtained will help assess the environmental safety associated with successive fertilization with antibiotics-contaminated manure in agricultural soil.
triplicate and incubated at 25 § 1 C in the dark. Five successive additions of SDZ and CTC were performed every 60 days. At the end of each treatment, 15.0 g of soil samples was collected from each plastic frame to determine soil microbial functional diversity. Sixty days after each treatment and 120 days after the final treatment, 10.0 g of soil samples was collected to determine bacterial community structure.
Materials and methods
DNA extraction and PCR-TGGE
Chemicals
The soil used in this study was collected from a mulberry field located at the Huajiachi Campus of Zhejiang University, Hangzhou, China. The soil had not been contaminated by SDZ and CTC. The soil samples were sieved (2 mm) to remove stones and debris. The soil texture was classified as a silt loam by its physicochemical properties (sand, 21.5%; silt, 71.1%; clay, 7.4%; organic matter content, 3.05%; water holding capacity, 39.4%; cationic exchange capacity, 10.6 cmol kg–1; total nitrogen, 0.14%; and pH 6.8). The pig manure was collected from a piggery located at the Huajiachi Campus of Zhejiang University, and manure chemical properties were determined as follows: soluble organic matter, 385.4 g kg–1; total nitrogen, 53.4 g kg–1; P2O5, 27.6 g kg–1; and pH 5.9.
Total soil DNA was extracted from each sample with a FastDNA Spin Kit according to the manufacturer’s protocol (Qbiogene, Carlsbad, CA, USA) and was then used as a template to amplify the 16S rDNA V3 region using the primer set GC341F and 518R with a TPersonal Thermalcycler (Whatman Biometra, Gottingen, Germany). PCR was conducted according to the method of Wang et al.[15] PCR products were analyzed by 1.0% agarose gel electrophoresis with TAE buffer containing 1.0 mg mL–1 ethidium bromide. The temperature gradient gel electrophoresis (TGGE) Mini system (Biometra, Gottingen, Germany) was used for sequence-specific separation of PCR products. Polyacrylamide gels were composed of 8% acrylamide/bis (37.5:1), 8 mol L–1 urea, 20% formamide and 2% glycerol. The electrophoresis was performed at a constant voltage of 200 V for 3 h in 1 £ TAE buffer (40 mmol L–1 Tris-Acetate, pH 8.0). A temperature gradient of 42 C to 50 C in parallel with the direction of electrophoresis was used. After the electrophoresis, gels were stained with AgNO3 as described by the manufacturer (Biometra). The stained gels were immediately photographed using a digital camera DSC-F717 (Sony, Tokyo, Japan).
Soil treatment and sampling
Phylogenetic analysis
Pig manure (80 g) was first suspended in sterile water, and SDZ and CTC in methanol solutions were successively added separately and together. Next, the pig manure was thoroughly mixed with the soil (2.0 kg dry weight equivalent) to obtain a normal agricultural manure amount of 4% (w/w) and a final antibiotic concentration of 20.0 mg kg–1 of dry soil. Finally, the soil mixtures were sieved (2-mm mesh) and transferred to a 25-L plastic frame covered with aluminum foil. Soil samples amended with the same volume water were used as untreated control. Soil water content was adjusted to 60% of the maximum water holding capacity and maintained by periodic distilled water additions. All treatments were carried out in
The specific bands on TGGE gels were cut out with sterile razor blades and eluted in 30 mL of sterile double-deionized H2O overnight at 4 C. Approximately 3 mL of elution was used as a PCR template under the conditions described above. The purified PCR products were ligated into a pMD19-T vector (TaKaRa, Dalian, China) according to the manufacturer’s recommendations, transformed into E. coli DH5a and then sequenced (Invitrogen, Shanghai, China). The obtained 16S rDNA sequences were submitted to the NCBI GenBank to identify the nearest genera or species according to the BLAST program. A phylogenetic tree of these sequences was constructed by MEGA5.05, with Thermocrinis ruber DSM 12173 used as the outgroup.[16]
Technical grade SDZ (purity 98.5%) and CTC (purity 98.5%) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Soils and manure
Carbon substrate utilization Carbon substrate utilization by soil microorganisms during repeated antibiotic treatments was determined according to the method described by Fang et al.[14]
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Statistical analysis The absorbance values at 72 h were used to calculate the Simpson diversity index (1/D), Shannon-Wiener index (H0 ), and McIntosh index (U) according to Eqs. (1)–(3): DD
X ðniðni ¡ 1ÞÞ
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ðN ðN ¡ 1ÞÞ X 0 H D¡ piðlnpiÞ rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi X ffi UD ni2
(1) (2) (3)
where ni is the absorbance value, N is the total absorbance value of all wells, pi is the proportional absorbance value of the well over the total absorbance value of all wells, and the Simpson index is the reciprocal (1/D). The 1/D, H0 , and U indices are used to assess the dominant population, richness, and evenness of soil microorganisms, respectively. Diversity index differences between different treatments were analyzed by one-way ANOVA using SPSS 11.5 software (SPSS Inc., Chicago, IL, USA).
Results Substrate utilization patterns of soil microorganisms The average well color development (AWCD) in the BIOLOG ECO plate, reflecting the oxidative capacity of soil microbial communities, usually serves as an indicator of overall microbial activities. As shown in Fig. 1, in the first 60-day treatment, the antibiotics treatment AWCD values were lower than the individual manure treatment values. In the second 60-day treatment, there was no significant difference observed in the AWCD values among all treatments. However, in the third, fourth, and fifth 60day treatments, the antibiotics treatment AWCD values were higher than those of the individual manure treatment. ANOVA showed that both the CTC treatment and SDZ C CTC treatment significantly (P 0.05) enhanced the AWCD values of soil microbial communities after the third treatment. However, no significant difference in the AWCD values was found between the individual manure treatment and SDZ treatment. Response of soil microbial functional diversity to repeated treatments The effects of repeated SDZ and CTC treatments on soil microbial functional diversity are summarized in Table 1. In the first 60-day treatment, the 1/D, H0 , and U indices decreased by 27.0%, 12.5%, and 28.2% in the SDZ treatment, 30.3%, 14.9%, and 40.8% in the CTC treatment, and 41.5%, 18.0%, and 29.6% in the SDZ C CTC treatment, respectively, compared with those in the
Fig. 1. AWCD variations throughout the incubation period for soil microbial communities during repeated treatments of SDZ and/or CTC.
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Table 1 Effects of repeated treatments of SDZ and/or CTC treatments on the functional diversity of soil microorganisms.
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Treatment frequency 1st 1st 1st 1st 1st 2nd 2nd 2nd 2nd 2nd 3rd 3rd 3rd 3rd 3rd 4th 4th 4th 4th 4th 5th 5th 5th 5th 5th
Time (days)
Treatment
Simpson index (1/D)
Shannon-Wiener index (H0 )
McIntosh index (U)
60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60
CK Manure SDZ CTC SDZCCTC CK Manure SDZ CTC SDZCCTC CK Manure SDZ CTC SDZCCTC CK Manure SDZ CTC SDZCCTC CK Manure SDZ CTC SDZCCTC
8.16 § 0.72 c 15.27 § 1.31 a 11.14 § 0.97 b 10.65 § 0.46 b 8.93 § 0.33 c 4.98 § 0.45 c 8.71 § 0.85 a 7.64 § 0.53 b 7.80 § 0.74 b 8.04 § 0.87 ab 6.62 § 0.45 e 7.52 § 0.74 d 9.43 § 0.45 c 10.41 § 1.02 b 12.43 § 1.83 a 9.26 § 0.43 b 9.34 § 0.83 b 10.13 § 0.98 a 10.51 § 0.52 a 10.76 § 0.99 a 5.99 § 0.75 e 6.07 § 0.45 d 8.04 § 0.46 c 9.55 § 0.74 b 11.07 § 1.02 a
2.23 § 0.08 b 2.89 § 0.08 a 2.53 § 0.06 ab 2.46 § 0.07 ab 2.37 § 0.06 ab 1.90 § 0.07 b 2.47 § 0.08 a 1.92 § 0.08 b 2.36 § 0.05 a 2.45 § 0.08 a 2.18 § 0.05 b 2.25 § 0.08 ab 2.41 § 0.06 a 2.62 § 0.07 a 2.68 § 0.04 a 2.39 § 0.08 a 2.41 § 0.07 a 2.48 § 0.07 a 2.61 § 0.05 a 2.61 § 0.08 a 2.08 § 0.08 b 2.02 § 0.08 b 2.42 § 0.05 a 2.50 § 0.07 a 2.65 § 0.09 a
1.10 § 0.09 b 2.13 § 0.24 a 1.53 § 0.07 b 1.26 § 0.08 b 1.50 § 0.10 b 0.89 § 0.07 b 1.94 § 0.08 a 1.08 § 0.04 b 1.27 § 0.07 b 0.97 § 0.09 b 1.63 § 0.12 c 1.37 § 0.05 c 1.45 § 0.08 c 2.96 § 0.15 a 2.14 § 0.10 b 1.42 § 0.09 b 1.59 § 0.12 b 1.84 § 0.10 b 1.78 § 0.11 b 2.51 § 0.16 a 2.21 § 0.10 a 1.31 § 0.09 b 2.02 § 0.12 a 2.74 § 0.15 a 2.58 § 0.20 a
Data followed by a different letter in the same column are significantly different (P 0.05).
individual manure treatment. In the second 60-day treatment, the 1/D and H0 indices in the antibiotic-treated soils showed a recovery, but the U index was still significantly smaller compared with individual manure treatment levels. The 1/D index in the SDZ, CTC, SDZCCTC treatments significantly (P 0.05) increased by 25.4%, 38.4%, and 65.3% in the third treatment, 8.5%, 12.5%, and 15.2% in the fourth treatment, and 32.5%, 57.3%, and 82.4% in the fifth treatment, respectively, and the H0 and U indices also slightly exceeded those in the individual manure treatment.
As shown in Fig. 2, the PC1 component (49.0%) in the PCA profile showed more important power than the PC2 component (13.7%). In the first 60-day treatment, a dispersed pattern was observed, and antibiotic treatment scores in the PC1 axis were lower than those in the individual manure treatment. This dispersed pattern was retained in the second and third 60-day treatments, and the scores of the antibiotic treatments in the PC1 axis were similar to those of the individual manure treatment. In the fourth and fifth 60-day treatments, a grouped pattern was gradually formed.
Response of soil microbial community structure to repeated treatments
Fig. 2. PCA analysis of soil microbial communities during the five repeated treatments of SDZ and/or CTC.
The TGGE profiles of 16S rDNA V3 regions derived from all treatments are shown in Fig. 3. Throughout this study, no obvious changes in the TGGE banding patterns were observed in the unamended control. During the five repeated treatments, more bands were detected in the manured soils than in the control soils. Furthermore, some specific bands were found between the individual manure soil and the antibiotics-treated soil. As shown in Fig. 3, specific bands 1–11 were selected, cut, and sequenced to identify soil microbial populations (the sequences of the 11 bands are summarized in Table S1). The
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Fig. 3. TGGE analysis of 16S rDNA fragments derived from the five repeated treatments of SDZ and/or CTC. Lanes 1–5: CK, Manure, SDZ, CTC, and SDZCCTC.
phylogeneticrelationshipbetweenthese11sequencesandreference sequences obtained from NCBI GenBank is presented in Fig. 4, and their phylogenetic affiliations are summarized in Table 2. In the first 60-day treatment, the dominant band 11 belonged to the phylum Actinobacteria and showed a high similarity (100%) to Rhodococcus equi strain DSM 20307. In
Fig. 4. Neighbor-joining tree showing the phylogenetic affiliations of 16S rDNA V3 sequences obtained from bands excised from TGGE gels during five repeated treatments of SDZ and/or CTC. The tree was rooted with the partial 16S rDNA sequence of Thermocrinis ruber DSM 12173 as an outgroup. Values at nodes represent the percentage of 1,000 bootstrap replicates. The scale bar indicates an estimated change of 5%.
the second 60-day treatment, the intensity of band 9 was much higher in the antibiotics-treated soils than in the control soils, and it belonged to the phylum Firmicutes and showed a high similarity (100%) to Clostridium disporicum strain DS1. Band10intheSDZtreatmentbelongedtothephylumActinobacteria and showed 97% similarity to Microlunatus aurantiacus strain YIM 45721, but it was not found in the CTC and SDZCCTC treatments. In the third 60-day treatment, the intensity of band 2 in the antibiotic treatments was higher than in the other treatments, which belonged to the phylum Proteobacteriaandshowed98%similaritytoSulfurovumlithotrophicum stain 42BKT. In the fourth 60-day treatment, band 3 was observed only in the antibiotic treatments affiliated with the phylum Firmicutes and showed 99% similarity to Dehalogenimonas lykanthroporepellens strain BL-DC-9. Inthefifth60-daytreatment,dominantbands5and8,belonging to the phyla Firmicutes and Proteobacteria, specifically formed in the antibiotic treatments. The intensity of band 7, belonging to Tenericutes, was much higher in the fifth treatment than in previous treatments, but it was not found in the CTCtreatment. In the 120 days after the final treatment, the intensities of bands 3, 4, and 9 (all of them belonging to the phylum Firmicutes) became lower than those in the five previous treatments. However, bands 5, 7, and 8 were still retained with high intensities in the TGGE profiles.
Discussion The decreased AWCD values from antibiotics-treated soils during the first treatment may be due to the suppression of bacteria sensitive to SDZ or CTC. In agreement with this result, Liu et al.[11] reported that six types of antibiotics (CTC, tetracycline, tylosin, sulfamethoxazole,
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Table 2 Phylogenetic affiliations of 16S rDNA V3 sequences excised from TGGE gels during five repeated treatments of SDZ and/or CTC. Band no.
Fragment length (bp)
Band 1 Band 2 Band 3 Band 4 Band 5 Band 6 Band 7 Band 8 Band 9 Band 10 Band 11
172 170 170 195 170 171 170 172 170 175 175
Closest relative in GenBank Sphaerobacter thermophilus strain DSM 20745 Sulfurovum lithotrophicum strain 42BKT Dehalogenimonas lykanthroporepellens strain BL-DC-9 Bacillus niabensis strain 4T19 Clostridium quinii strain DSM 6736 Clostridium cellulovorans strain 743B Mycoplasma glycophilum strain 486 Anaerobiospirillum succiniciproducens strain S411 Clostridium disporicum strain DS1 Microlunatus aurantiacus strain YIM 45721 Rhodococcus equi strain DSM 20307
sulfamethazine, and trimethoprim) could suppress soil microbial activities. Girardi et al.[17] revealed that ciprofloxacin strongly inhibited microbial activities. Liu et al.[18] reported that 100 mg kg–1 of sulfamethoxazole had a significant inhibitory effect on AWCD values after a 7-day treatment. In this study, however, the significant increase of AWCD values in CTC and SDZCCTC treatments in the third treatment indicated that CTC might have a stimulatory effect on soil microbial activities, possibly due to the formation and proliferation of tolerant or resistant bacteria. A similar finding was also reported by Liu et al.[19] that oxytetracycline could increase the AWCD values in a 7-week greenhouse pot experiment with BIOLOG ECO microplates. In this study, the AWCD values from the manured soils were much higher than those from the unamended control soils. This phenomenon is not surprising because manure application could increase the soil nutrition and lead to a microbial biomass increase.[20,21] During the first treatment, the 1/D, H0 , and U indices decreased significantly (P 0.05) in the antibiotic treatments, which indicated that the dominant populations, richness, and evenness of soil microorganisms were suppressed by SDZ or CTC. Similar to this finding, Kong et al.[22] reported that the H0 and U indices decreased significantly (P 0.005) with increasing concentrations of oxytetracycline. Liu et al.[18] reported that the H0 index could be suppressed by 100 mg kg–1 of sulfamethoxazole after a 7-day treatment. In contrast, Muller et al.[23] revealed no H0 index differences in tylosin-treated soils. PCA analysis showed a trend from dispersed patterns to grouped patterns, indicating an initial suppression of sensitive bacteria by antibiotics, but then, repeated treatments stimulated growth of resistant bacteria and improved microbial activities. As shown in the TGGE profiles, the total number of bands in the manure treatment was considerably higher than in the manure-unamended treatment. A similar phenomenon was reported by Hammesfahr et al.[10,20] who showed that manure had a stimulating effect on soil microbial communities such as PLFAtot and could increase band
Similarity
Putative phylum
99% 98% 99% 100% 98% 87% 94% 88% 100% 97% 100%
Actinobacteria Proteobacteria Firmicutes Firmicutes Firmicutes Firmicutes Tenericutes Proteobacteria Firmicutes Actinobacteria Actinobacteria
intensity. In this study, repeated treatments of SDZ and/ or CTC altered soil microbial community structure. Hammesfahr et al.[10] reported that a single application of SDZ in combination with manure had a prolonged effect on the microbial community structure, resulting in altered community structure. In this study, band 10 occurred in the SDZ treatment but was not found in the CTC and SDZCCTC treatments, suggesting that CTC has an inhibitory effect on such bacteria. The high intensity of bands 2, 3, 5, and 8 in the antibiotic treatments indicated that SDZ or CTC caused the formation and proliferation of these resistant bacteria. Band 7 occurred in the SDZ and SDZCCTC treatments but was not found in the CTC treatment, indicating that SDZ may have a stimulatory effect on these microbial species. Some studies reported that SDZ treatment induced the formation of additional bands, representing related resistant bacteria.[12,24] Hammesfahr et al.[20] reported that microbial structure was more sensitive to SDZ contamination than functional processes, and the amendment of pig liquid manure and SDZ altered microbial community structure. Furthermore, the effect of SDZ on microbial community structure increased with incremental liquid manure amendment. The findings in this study indicate that the functional diversity of the soil microbial community was temporarily inhibited during the first treatment but gradually recovered with further treatments. Repeated treatments of SDZ and/ or CTC altered soil microbial community structure. Therefore, repeated treatments of antibiotics may have a persistent impact on soil health. Further studies need to be conducted to reveal the diversity, abundance, and expression level of resistant genes in the antibiotic-contaminated soils.
Funding This work was supported by the National High Technology R&D Program of China (No. 2012AA06A204), the
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Microbial response to repeated treatments of manure National Nature Science Foundation of China (No. 21377112, 20907040), and the Public Science and Technology Research Funds Projects of the Ministry of Environmental Protection of China (No. 201109018).
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Supplemental Material Supplemental data for this article can be accessed on the publisher’s website.
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