Occurrence of Chloramphenicol-Resistance Genes as Environmental

Feb 18, 2013 - Chloramphenicol-resistance genes could be propagated to the surrounding environment via agricultural application of swine waste. This s...
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Occurrence of Chloramphenicol-Resistance Genes as Environmental Pollutants from Swine Feedlots Juan Li,†,‡,§ Bing Shao,§,∥ Jianzhong Shen,† Shaochen Wang,‡,⊥ and Yongning Wu†,‡,⊥,* †

Key Laboratory of Development and Evaluation of Chemical and Herbal Drugs for Animal Use, College of Veterinary Medicine, China Agricultural University, Beijing 100193 ‡ Key Laboratory of Food Safety Risk Assessment, Ministry of Health, China National Center for Food Safety Risk Assessment, Beijing 100021 § Beijing Key Laboratory of Food Poison Diagnostic and Traceability, Beijing Center for Disease Control and Prevention, Beijing 100013 ∥ College of Public Health and Family Medicine, Capital Medical University, Beijing 100069 ⊥ Key Laboratory of Chemical Safety and Health, Chinese Center for Disease Control and Prevention, Beijing 100050 S Supporting Information *

ABSTRACT: Chloramphenicol-resistance genes could be propagated to the surrounding environment via agricultural application of swine waste. This study investigated the potential risks of chloramphenicol-resistance genes from swine feedlots and their surrounding environment. We applied a culture-independent method to investigate levels of chloramphenicol-resistance genes in the wastewater from swine feedlots and the correspondingly impacted agricultural fields in Beijing. The cmlA, f loR, fexA, cf r, and fexB genes were present in all samples, with the highest absolute concentrations of 1.50 × 106 copies/g in soil and 6.69 × 106 copies/mL in wastewater. The concentration of chloramphenicol residue was determined by ultra performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS), with the highest concentrations of 0.83 ng/g in soil and 11.5 ng/mL in wastewater. Significant correlations were found between chloramphenicol-resistance genes and chloramphenicol residues (r = 0.79, p = 0.0008) as well as between chloramphenicol-resistance genes in swine feedlots and corresponding agricultural soils (r = 0.84, p = 0.02). Consequently, swine feedlot wastewater could become a source of chloramphenicol-resistance genes, which could then lead to the spread of antibiotic resistance and eventually pose a risk to public health. To our knowledge, this is the first study to examine the occurrence of f loR, fexA, cf r, and fexB genes in the environment using a culture-independent method.

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INTRODUCTION

Antibiotic resistance genes (ARGs), released from dead microorganisms, have the potential to be transported over considerable distances in the environment.6 They could be subsequently spread in the environment by certain bacteria through vertical transfer or horizontal transfer. This could ultimately result in the ubiquitous propagation of antibiotic resistance and potential risks to the environment and human health. ARGs as emerging environmental contaminants7 have the potential to be widely distributed to various environmental compartments.8,9 Compared with traditional contaminants, they are heritable and could pose a long-term and irreversible risk to the environment and public health, which would be difficult to control.10 The culture-independent method refers to analysis of the organisms’ assemblage in environmental samples without culture of bacteria.11 This avoids the bias of the unculturability of a large proportion of environmental micro-

The use of chloramphenicol has been banned in foodproducing animals in the European Union (EU) since 1994 because of its adverse side-effects.1 However, sometimes, chloramphenicol might be still illicitly used by some small farmers, since it is inexpensive, broad-spectrum, and easy to store.2 Its use in animals was significantly associated with increased chance of resistance to chloramphenicol in bacteria.3,4 There are three main mechanisms responsible for resistance to chloramphenicols:3 chloramphenicol acetyltransferases (encoded by cat genes); chloramphenicol export mediated by multidrug transporters or specific transporters (encoded at least by cmlA, f loR, fexA, pexA, and fexB genes); and rRNA methylase mediated by the cfr gene, which simultaneously confers resistance to chloramphenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A antibiotics.5 These genes reside on mobile genetic elements, such as plasmids, transposons, or integrons.3 They might have paved the way for a distribution of chloramphenicol-resistance genes across species and genus borders. © 2013 American Chemical Society

Received: Revised: Accepted: Published: 2892

November 14, 2012 February 5, 2013 February 18, 2013 February 18, 2013 dx.doi.org/10.1021/es304616c | Environ. Sci. Technol. 2013, 47, 2892−2897

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organisms in standard culture conditions12 and the difficulty of making conclusive comparisons among various environmental media.13 The method has been used to investigate plasmidmediated quinolone resistance (PMQR) genes in environmental samples.14 Swine waste containing resistance genes and unabsorbed antibiotics has typically been stored in open-air lagoons and subsequently applied to surrounding agriculture fields through irrigation or fertilization and even poured into surrounding waterways through ditches.14,15 As a result of various natural factors such as runoff or wind, they affect broad environmental compartments and may eventual affect human settlements risking public health.7 Various resistance genes have been found in environmental compartments influenced by surrounding swine feedlots.14,16,17 To our knowledge, however, few investigations have been carried out on the chloramphenicolresistance genes (especially for f loR, fexA, cf r, and fexB) in the environment of animal husbandry. This is the first study, using a culture-independent method, to investigate the occurrence of the typical chloramphenicol-resistance genes in wastewater samples gathered from different swine feedlots and soil samples collected from corresponding agricultural fields around three districts of Beijing, China.

CA, USA) with Agilent DNA 7500 LabChip kit (Agilent Technologies) was used to analyze DNA fragments.18 Amplification products from each positive sample were cloned into Escherichia coli (E.coli) DH5α. Clones containing target gene inserts were extracted and purified with a Plasmid Kit (TaKaRa, Dalian, China) and sequenced by Invitrogen Ltd./ Applied Biosystems Ltd. (Beijing, China). These plasmids were used to generate standard curves for subsequent quantification of each gene. Sequences were compared with GenBank sequences for the target genes using the BLAST alignment tool (http://www.ncbi.nlm.nih.gov/blast/). The identity of results are described in the Supporting Information. For qPCR reactions, the quantification methods were described previously14 (details in the Supporting Information). Simply, amplifications of chloramphenicol-resistance genes were conducted in a 20 μL reaction volume containing 1 μL template DNA, 0.2 μL 10 nmol/L of each primer, MightyAmp Real Time (SYBR Plus) mix (TaKaRa, China) and molecular biology-grade water using a Bio-Rad Chromo4 real-time PCR instrument (Bio-Rad, U.S.) with the Analysis software version 3.0 (BioRad, U.S.). The thermal cycle was determined empirically, consisting of 10 min initial denaturation at 95 °C, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing for 45 s at different temperatures (Table S1 of the Supporting Information), then 72 °C for 1 min, with reading plate after each cycle. 16S rRNA was in the same reaction mixtures as the chloramphenicol-resistance genes, with a temperature program of 15 min at 95 °C, followed by 40 cycles of the following: 20 s at 95 °C, 45 s at 62 °C, and 30 s at 72 °C, with reading plate after each cycle. Melting curves for the amplicons were measured by raising the temperature slowly while monitoring fluorescence to verify that nonspecific amplification did not occur (data not shown). In this study, we defined the copies of each chloramphenicolresistance gene normalized to 1 g of soil samples or 1 mL of wastewater samples as the absolute concentration (copies/g or copies/mL). Moreover, to take into account any temporal variations among stations, overall extraction efficiencies, total bacterial community, and possible sample degradation,19 the copies of each chloramphenicol-resistance gene were normalized to the 16S rRNA copy number (defined as relative abundance). We used the term levels to describe findings that relate to both relative abundances and the absolute concentrations. Quantitation of Chloramphenicol. In this study, extraction and quantitation of chloramphenicol in wastewater, soil and control samples have been described previously14,20 with some modifications using solid phase extraction and ultra performance liquid chromatography-tandem mass spectrometry (SPE-UPLC-MS/MS) on a Waters Acquity Ultra Performance LC system (Waters, Milford, MA, USA) with the software of MassLynx (detailed in the Supporting Information). The recoveries for chloramphenicol based on matrix-matched calibration were 77% in wastewater samples and 72% in soil samples, whereas the quantification limits were 3 pg/mL for water and 0.2 ng/g for soil. Statistical Methods. In this study, SPSS version 16.0 was used to perform data analysis. The homogeneity of values was assessed via a one-way analysis of variance (ANOVA) test (significant at p < 0.05 level). And linkages were assessed by a two-tailed Pearson’s bivariate correlation analysis, between chloramphenicol-resistance genes in soil and wastewater

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EXPERIMENTAL SECTION Sample Collection and Preparation. In the study, the detailed methods of sample collection and preparation were described previously.14 Wastewater and soil samples were respectively collected from the effluent of and adjacent to seven conventional swine feedlots located in three districts of Beijing; Fangshan District (defined as F1-w, F2-w, and F3-w for wastewater samples; F1-s, F2-s, and F3-s for soil samples), Daxing District (D1-w, D2-w, and D3-w for wastewater samples; D1-s, D2-s, and D3-s for soil samples), and Shunyi District (S-w for wastewater samples and S-s for soil samples) during August 2010. Therefore, F1-w and F1-s are paired wastewater and soil samples from the same site, and this also applies for the other sample sites. For the seven conventional swine feedlots, effluents are periodically used for irrigation in surrounding agricultural fields and occasionally discharged into surrounding rivers. In addition, control samples, including water and soil, were respectively collected in surface river water and corresponding farm at sites upstream from the swine feedlots (defined as being uncontaminated by wastewater from swine feedlot operations). DNA Extraction. Individual wastewater samples (about 200 mL) were used to extract total DNA using the Power Water DNA Kit (MO BIO Laboratories Inc., Carlsbad, CA, USA) following the manufacturer’s protocol. About 1 g of homogeneous soil was extracted using a commercial Power Soil DNA Kit (MO BIO) according to the manufacturer’s instructions. The methods have been described previously.14 PCR and QPCR Assays. 16S rRNA and some typical chloramphenicol-resistance genes (cmlA, f loR, fexA, cfr, and fexB) were investigated and enumerated using qualitative PCR and quantitative PCR (qPCR) in the environmental samples and the control samples, respectively. All primers for these genes were either reported previously or newly designed (Table S1 of the Supporting Information). In PCR amplification, the procedure was detailed in the Supporting Information. Two replicates for each sample were performed in parallel with a control sample in each run. An Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, 2893

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Figure 1. Levels of chloramphenicol-resistance genes among the samples. (A) cmlA, (B) f loR, (C) fexA, (D) cf r, (E) fexB, (F) total of the five chloramphenicol-resistance genes (cmlA, f loR, fexA, cf r, and fexB). Bars indicate the means of absolute concentrations, whereas symbols indicate the means of relative abundances. Black bars and symbols indicate values in wastewater samples, while gray bars and blank symbols indicate values in soil samples. Codes on x axis indicate the sample sites as given in the Experimental section. Error bar represents standard error of three replicates.

Table 1. Correlationsa between Chloramphenicol-Resistance Genes in Wastewater and Soil Samples absolute concentration relative abundanced

c

cmlA

f loR

fexA

cf r

fexB

totalb

0.27 (0.56) 0.90 (0.006)

−0.02 (0.97) −0.63 (0.13)

0.88 (0.009) 0.97 (0.0003)

−0.05 (0.92) 0.63 (0.13)

−0.41 (0.36) 0.52 (0.23)

−0.05 (0.92) 0.84 (0.02)

a

Values indicate the Pearson correlation coefficient (r), and the p-values are presented in brackets. The bold values in italics indicate statistical significance (p < 0.05). bSum of five chloramphenicol-resistance genes (cmlA, f loR, fexA, cf r, and fexB). cThe absolute copies of chloramphenicolresistance genes normalized to sample volume or mass. dThe absolute copies of chloramphenicol-resistance genes normalized to corresponding 16S rRNA copies.

Tables S2 and S3 of the Supporting Information summarize the absolute concentrations and relative abundances of the five genes (cmlA, floR, fexA, cf r, and fexB), ranging of 3.30 × 104 to 1.63 × 106 copies/mL and 7.01 × 10−3 to 4.72 × 10−2 for wastewater samples and of 4.90 × 104 to 4.94 × 105 copies/g and 4.52 × 10−3 to 3.17 × 10−2 for soil samples, respectively. Quantification of Chloramphenicol in Samples. Although the use of chloramphenicol has been banned in food-producing animals in China,20 chloramphenicol residue was commonly detected in all environment samples, except for the D1-s and D3-s samples in which the concentrations were below the detection limit of the UPLC−MS/MS method in this study. The concentration of chloramphenicol was below the detection limit in all control samples. This result probably reflects its illicit use in swine feeding practices and its poor absorption by pigs.17,20 The chloramphenicol residue average concentrations ranged from 1.49 to 11.5 ng/mL in wastewater samples and from below the detection limit to 0.83 ng/g in soil samples. Among the concentrations of both wastewater samples and soil samples, the highest concentrations were observed in the D2-w sample (11.5 ng/mL) and the F3-s sample (0.83 ng/ g), respectively. The variation of concentrations among these swine feedlots was within the same order of magnitude, which might reveal the little difference in chloramphenicol usage among these swine feedlots. Responses of Swine Feedlot Effluents to Surrounding Agricultural Fields. The distribution of chloramphenicolresistance genes among these environment samples are

samples, as well as between chloramphenicol-resistance genes and chloramphenicol residues.

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RESULTS AND DISCUSSION Chloramphenicol-Resistance Genes in Samples. In this study, cmlA, f loR, fexA, cf r, and fexB were observed in all environmental samples whereas none of them were detected in the control samples (Figure S1 of the Supporting Information). It was not surprising. The resistance to chloramphenicols has become widespread and serious. The five chloramphenicolresistance genes usually reside on mobile genetic elements, which most often carry additional resistance genes and virulence genes and the coselection and persistence of them might occur even in the absence of selection pressure.3 To date, among the known chloramphenicol-resistance genes, several types of cml genes have been reported to be of environmental origin21 and these five genes (cmlA, f loR, fexA, cf r, and fexB) have been shown as localized on mobile genetic elements in several bacterial strains from swine,22−25 especially the novel phenicol exporter gene fexB, which was reported recently on plasmids of E. faecium and E. hirae.26 Interestingly, the cat genes (including cat I, catII, catIII and catIV) which have been reported commonly in the environment of China27−29 were absent in all samples in this study. Therefore, the occurrence of chloramphenicol-resistance genes might vary depending on sampling sites, and this presumption is similar to results from by Cummings et al.30 regarding PMQR genes. 2894

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Table 2. Correlationsa between Chloramphenicol-Resistance Genes and Chloramphenicol Residues absolute concentration relative abundanced

c

cmlA

f loR

fexA

cf r

fexB

totalb

0.72 (0.004) 0.50 (0.07)

0.80 (0.0005) 0.58 (0.03)

0.81 (0.0004) 0.81 (0.0004)

0.70 (0.006) 0.45 (0.11)

0.78 (0.001) 0.66 (0.01)

0.79 (0.0007) 0.79 (0.0008)

a

Values indicate the Pearson correlation coefficient (r), and the p-values are presented in brackets. The bold values in italics indicate statistical significance (p < 0.05). bSum of five chloramphenicol-resistance genes (cmlA, f loR, fexA, cf r, and fexB). cThe absolute copies of chloramphenicolresistance genes normalized to sample volume or mass. dThe absolute copies of chloramphenicol-resistance genes normalized to corresponding 16S rRNA copies.

Response of Chloramphenicol to ChloramphenicolResistance Genes. Moderate significant correlations were exhibited between chloramphenicol and individual chloramphenicol-resistance gene absolute concentration among environmental samples collected from all detected swine feedlots (Table 2; cmlA: r = 0.72, p = 0.004; f loR: r = 0.80, p = 0.0005; fexA: r = 0.81, p = 0.0004; cfr: r = 0.70, p = 0.006; fexB: r = 0.78, p = 0.001). Additionally, there were moderately significant correlations between chloramphenicol and total chloramphenicol-resistance genes with regard to both absolute concentration (Table 2 and Figure S3 of the Supporting Information; r = 0.79, p = 0.0007) and relative abundance (Table 2 and Figure S3 of the Supporting Information; r = 0.79, p = 0.0008). These observations are similar to previous results14 and consistent with the prior hypothesis that constant exposure to antibiotics would lead to selective pressure for resistance genes.17 However, some of these relationships were not as strong as those found in other studies,37 which could be the result of different environmental fate and transport mechanisms of resistance genes versus antibiotics after their release.38 Up to now, numerous of studies were conducted to investigate the changes of microbial communities due to the antibiotic residues in soil and water environment.39 It was shown that antibiotic residues in environment could alter the microbial community structure,40 inhibit or promote ecological functions,41 and may affect the selection and dissemination of resistance.42 However, how exactly the low concentration of antibiotic residues affects the microbial resistance, particularly for low-level chloramphenicol residue in environment, remains unknown and further research in the area is needed in the future. To our knowledge, this is the first study to examine the chloramphenicol-resistance genes in piggery settings using a culture-independent method with f loR, fexA, cf r, and fexB genes reported in environmental samples. Furthermore, the chloramphenicol-resistance genes in fields adjacent to swine feedlots were demonstrated to have most likely originated from these piggeries through waste amendment, highlighting the role of swine feedlot as source of antibiotic resistance genes to other environment compartments. Correlation between total chloramphenicol-resistance genes with chloramphenicol residues further signifies that environmental antibiotic residues might give rise to the positive selection of antibiotic resistance genes. Overall, considering the rapid expansion of piggery industries and the seriousness and potential risks of chloramphenicolresistance genes to human health, it is urgently recommended that surveillance programs for the presence of various antibiotic resistance genes in the environment on a global scale should be established.

displayed in Figure 1. Compared with previous data, the levels of chloramphenicol-resistance genes were lower than levels of PMQR genes both in soil and wastewater samples.14 There was a different degree of pollution in the same samples for different kinds of antibiotic resistance gene contaminants. Pearson correlation analyses were conducted to investigate potential relationships between soil and wastewater samples with regard to the levels of chloramphenicol-resistance genes and the concentration of chloramphenicol residue. Table 1 illustrates a strong positive correlation between wastewater and soil samples with regard to the relative abundances of cmlA (r = 0.90, p = 0.006) and fexA (r = 0.97, p = 0.0003), whereas no significant correlations were observed for f loR (r = −0.63, p = 0.13), cf r (r = 0.63, p = 0.13) and fexB (r = 0.52, p = 0.23). On the contrary, there was no positive correlation between wastewater and soil samples in respect to the absolute concentrations of each and all of the chloramphenicol-resistance genes, except for the fexA (r = 0.88, p = 0.009). Nevertheless, a strong positive correlation was observed for total chloramphenicol-resistance genes (sum of five chloramphenicol-resistance genes: cmlA, f loR, fexA, cf r, and fexB) on the relative abundance (r = 0.84, p = 0.02) (Table 1 and Figure S2 of the Supporting Information). These further illustrate that swine feedlot wastewater as a source of chloramphenicol-resistance genes could carry these genes from swine feedlots to agricultural fields during agricultural application of swine waste and wastewater. Intriguingly, no significant correlation was found between the concentrations of wastewater and soil samples for chloramphenicol residue (data not shown). This may reflect the degradation of chloramphenicol due to environmental factors such as photodegradation, microbial degradation and plant adsorption, as suggested by Parshikov et al.31 and Torniainen et al.32 The swine wastewater containing chloramphenicol-resistance genes and chloramphenicol residue applied to proximal agricultural fields and released to the surrounding rivers through ditches may pose health risks to nearby residents who either are exposed to contaminated field soil during farming practices or use contaminated river water for living purposes. We have presented this hypothesis previously.14 Also, this has been confirmed in previous studies which suggested that there is a high risk for swine farmers, veterinarians, or people with exposure to swine farming to be colonized with some strains,33,34 especially by Kehrenberg et al.,35 who detected cf r genes in isolates of both clonal lineages, suggesting a possible spread to humans with exposure to swine farming. Sapkota et al.15 also found that the presence of resistant bacteria in surface water sources contaminated by swine waste could contribute to the spread of antibiotic resistance determinants in humans and the environment. In addition, broad use of antimicrobials in agriculture could select for resistant bacteria that may enter the food chain and potentially result in food-borne illness in humans that is less responsive to treatment with conventional antibiotics.36 2895

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ASSOCIATED CONTENT

S Supporting Information *

QPCR and PCR primers and amplification conditions, absolute concentrations and relative abundances of chloramphenicolresistance genes, typical Agilent 2100 bioanalyzer gel images of amplified chloramphenicol-resistance genes, correlations of chloramphenicol-resistance genes between paired wastewater and soil samples, as well as between chloramphenicol-resistance genes and chloramphenicol residue, five chloramphenicolresistance genes sequencing results, technical details of relevant methods as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]; Tel: 86-10-67779118; Fax: 86-10-67791253. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (No. 20837003), the National Basic Research Program of China (973 Program, No. 2012CB720804), and the Ministry of Health, PR China (No. 200902009).

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REFERENCES

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dx.doi.org/10.1021/es304616c | Environ. Sci. Technol. 2013, 47, 2892−2897