Persistent spread of the rmtB 16S rRNA methyltransferase gene among Escherichia coli isolates from diseased food-producing animals in China

Veterinary Microbiology 188 (2016) 41–46

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Persistent spread of the rmtB 16S rRNA methyltransferase gene among Escherichia coli isolates from diseased food-producing animals in China Jing Xiaa,b,1, Jian Suna,b,1, Ke Chenga,b , Liang Lia,b , Liang-Xing Fanga,b , Meng-Ting Zoua,b , Xiao-Ping Liaoa,b , Ya-Hong Liua,b,c,* a b c

National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, South China Agricultural University, Guangzhou, PR China College of Veterinary Medicine, South China Agricultural University, Guangzhou, PR China Jiangsu Co-Innovation Centre for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, PR China

A R T I C L E I N F O

Article history: Received 1 December 2015 Received in revised form 24 March 2016 Accepted 25 March 2016 Keywords: Escherichia coli rmtB 16S rRNA methyltransferase Food-producing animals IncF plasmids

A B S T R A C T

A total of 963 non-duplicate Escherichia coli strains isolated from food-producing animals between 2002 and 2012 were screened for the presence of the 16S rRNA methyltransferase genes. Among the positive isolates, resistance determinants to extended spectrum b-lactamases, plasmid-mediated quinolone resistance genes as well as floR and fosA/A3/C2 were detected using PCR analysis. These isolates were further subjected to antimicrobial susceptibility testing, molecular typing, PCR-based plasmid replicon typing and plasmid analysis. Of the 963 E. coli isolates, 173 (18.0%), 3 (0.3%) and 2 (0.2%) were rmtB-, armA- and rmtE-positive strains, respectively. All the 16S rRNA methyltransferase gene-positive isolates were multidrug resistant and over 90% of them carried one or more type of resistance gene. IncF (especially IncFII) and non-typeable plasmids played the main role in the dissemination of rmtB, followed by the IncN plasmids. Plasmids that harbored rmtB ranged in size from 20 kb to 340 kb EcoRI-RFLP testing of the 109 rmtB-positive plasmids from different years and different origins suggested that horizontal (among diverse animals) and vertical transfer of IncF, non-typeable and IncN-type plasmids were responsible for the spread of rmtB gene. In summary, our findings highlight that rmtB was the most prevalent 16S rRNA methyltransferase gene, which present persistent spread in food-producing animals in China and a diverse group of plasmids was responsible for rmtB dissemination. ã 2016 Elsevier B.V. All rights reserved.

1. Introduction Aminoglycoside antibiotics are widely used in clinical settings especially for the treatment of life-threatening infections caused by gram-negative bacteria (Doi et al., 2004). Members of this antibiotic class are active against a broad spectrum of target bacteria and have potent concentration-dependent bactericidal activity, post-antibiotic effects and have an ability to act synergistically with many other antibiotics. The widespread use of aminoglycosides in food-producing animals to prevent and control bacterial infections, as well as for growth promotion, has led to the emergence of widespread resistance to these drugs (Chen et al., 2007; Doi et al., 2004). In China, streptomycin, kanamycin, gentamicin, neomycin,

* Corresponding author at: College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, PR China. E-mail address: [email protected] (Y.-H. Liu). 1 These authors contributed equally to this article. http://dx.doi.org/10.1016/j.vetmic.2016.03.018 0378-1135/ ã 2016 Elsevier B.V. All rights reserved.

spectinomycin and apramycin have been approved to treat E. coli infections in food-producing animals. Moreover, several clinical first-line aminoglycoside agents such as neomycin and amikacin, are being misused in conventional broiler chicken and swine production facilities in China (Qin et al., 2012). The most frequently encountered resistance mechanisms to aminoglycosides are as follows: 1) structural modifications by specific enzymes; 2) mutation or modification of the aminoglycoside-binding site in the target molecule; 3) decreased permeability of bacterial membranes; and 4) augmented expression of efflux genes (Wachino and Arakawa, 2012). Recently, however, 16S rRNA methylases (16S-RMTase) have been shown to be responsible for high-level resistance against a range of aminoglycosides in Gram-negative bacilli. 16S rRNA methylases have emerged as a novel resistance mechanism to aminoglycosides (Wachino and Arakawa, 2012). Since the first report in 2003, ten 16S-RMTase encoding genes, rmtA, rmtB, rmtC, rmtD, rmtE, rmtF, rmtG, rmtH, armA and npmA, have been identified (O’Hara et al., 2013). These resistance determinants are globally disseminated, with rmtB and armA

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J. Xia et al. / Veterinary Microbiology 188 (2016) 41–46

being the most frequently reported. In addition, the 16S-RMTase genes, especially rmtB, are commonly associated with blaCTX-M, plasmid-mediated quinolone resistance (PMQR) and fosA3 genes (Deng et al., 2011a; Hou et al., 2012; Liu et al., 2008). Reports on the prevalence of the 16S-RMTases have increased in the past years with the majority focused on the original human clinical isolates of the Enterobacteriaceae (Wu et al., 2009; Yang et al., 2011). The few papers concerning 16S-RMTase prevalence in animals gave more attention to pets (Hou et al., 2012). Importantly, bacterial resistance to antimicrobials from food-producing animals has a considerable impact on human health (Zhang et al., 2012). Thus, our aim in this study was to investigate the prevalence and dissemination of 16S-RMTases of E. coli in food-producing animals in China, and to study their coexistence and co-transfer with other important resistance genes. 2. Materials and methods 2.1. Bacterial isolates and antimicrobial susceptibility testing A total of 963 non-duplicate E. coli strains isolated from foodproducing animals between 2002 and 2012 in Guangdong, China, were used in this study. Among them, 484 isolates from swine and 479 isolates from birds (126 from chicken and 353 from duck) were screened to understand the spread of 16S-RMTase genes in foodproducing animals. The isolates selected from more than 100 farms throughout Guangdong province (including the cities of Guangzhou, Zhaoqing, Yunfu, Qingyuan, Meizhou, Heyuan, Jiangmen, Foshan, Zhongshan, Shenzhen and Maoming) were recovered directly from farms and diagnostic laboratories. The fecal samples and swabs from animal organs (liver, heart or lung) were collected from diarrheal animals and from organs that exhibited pathological changes due to E. coli infection, respectively. Details of the E. coli isolates, included in this study are shown in Supplementary material Table S1 (a) (b) in the online version at DOI: 10.1016/j. vetmic.2016.03.018. Further information about these animals, their underlying disease and possible antimicrobial pretreatments were unfortunately not available. Cotton swabs of the liver, heart and lung tissues or feces from these animals were streaked on MacConkey agar without antibiotics. After 16 h incubation at 37
C, one colony with typical E. coli morphology from each sample was randomly selected and then identified by classical biochemical methods and the API 20E system (bioMerieux). All identified isolates were stored at 80
C in Luria-Bertani broth containing 30% glycerol. Amikacin-resistant E. coli was selected from the MacConkey agar containing amikacin at 64 mg/mL. Then 16S-RMTase genes were detected by a multiplex-PCR method previously developed (Corrêa et al., 2014). The minimal inhibitory concentrations (MICs) of antibiotics were determined by the agar dilution method according to the standards and guidelines described by the Clinical and Laboratory Standards Institute (CLSI, 2015: M100-S25) and veterinary CLSI (VET01-A4/VET01-S2). The following antimicrobial agents were assessed: ampicillin (AMP), cefotaxime (CTX), cefoxitin (FOX), meropenem (MEM), streptomycin (STR), amikacin (AMK), gentamicin (GEN), kanamycin (KAN), neomycin (NEO), apramycin (APR), chloramphenicol (CHL), florfenicol (FFC), tetracycline (TET), nalidixic acid (NAL), ciprofloxacin (CIP), norfloxacin (NOR), olaquindox (Oqx), trimethoprim-sulfamethoxazole (SMZ/TMP), colistin (CS) and fosfomycin (FOS). E. coli ATCC 25922 was used as a quality control strain.

sequencing. These isolates were further screened for the presence of the PMQR genes (qnrA, qnrB, qnrC, qnrD, qnrS, qepA, aac(60 )-Ib-cr and oqxAB) as well as floR and fosA/A3/C2. The PCR programs and primer sequences have been described previously (Hou et al., 2012; Li et al., 2013; Liao et al., 2015; Rao et al., 2014; Yang et al., 2015a; Zhang et al., 2015). The primers and PCR programs were listed in Table S2 (see Supplementary material Table S2 in the online version at DOI: 10.1016/j.vetmic.2016.03.018). 2.3. Molecular typing Genomic DNA of 16S-RMTase genes-positive isolates were analyzed by pulsed-field gel electrophoresis (PFGE) following digestion with XbaI (Shabana et al., 2013). Comparison of PFGE patterns was performed with BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium). Dendrograms were generated with the Dice similarity coefficient (1.5% optimization and 1.5% tolerance) using the unweighted pair-group method with arithmetic mean (UPGMA).PFGE types were defined with >90% similarity between clusters. 2.4. Transfer of the16S-RMTase genes and plasmid replicon typing Transferability of the identified 16S-RMTase genes was determined by conjugation using streptomycin-resistant E. coli C600 as the recipient strain (Chen et al., 2007). Transconjugants were selected on MacConkey agar plates supplemented with amikacin (64 mg/mL) and streptomycin (2000 mg/mL). Incompatibility (Inc) groups were assigned by PCR-based replicon typing of transconjugants (Fang et al., 2015). To better characterize IncF, replicon sequencing typing (RST) was performed according to protocols described previously, and alleles were assigned by comparing the amplicon sequence to the plasmid MLST database (http://pubmlst. org/plasmid/). Because of the multi-replicon nature of these plasmids, each plasmid can be identified using the FAB (FII, FIA, FIB) formula by the allele type and number identified for each replicon. The A- and B-symbols indicate the absence of the FIA and FIB replicons, respectively (Villa et al., 2010). Co-transfer of other resistance genes mentioned above was also determined using PCR. 2.5. Gene location and restriction fragment length polymorphism (RFLP) analysis PFGE using S1 nuclease (TakaRa Biotechnology, Dalian, China) digestion of whole genomic DNA was performed for all transconjugants as described previously (Barton et al., 1995). After Southern transfer to a Hybond-N+ membrane (GE Healthcare, Little Chalfont, United Kingdom), the plasmids were probed with

2.2. Resistance genes characterization

M,

All 16S-RMTase gene-positive isolates were screened for blaCTXblaTEM, blaSHV, blaDHA, blaCMY-1G and blaCMY-2G by PCR and DNA

Fig. 1. Prevalence of amikacin resistance and the rmtB gene among E. coli isolates.

J. Xia et al. / Veterinary Microbiology 188 (2016) 41–46

the 16S rRNA methyltransferase gene (Roche Diagnostics GmbH, Mannheim, Germany). Transconjugants containing one plasmid were extracted with a rapid alkaline lysis procedure and analyzed by RFLP using EcoRI digestion (Deng et al., 2011b). EcoRI-RFLP types were defined with >80% similarity between clusters using the method described for PFGE analysis. 2.6. Statistical analysis Statistical significance for the comparison of prevalence data and proportions was determined by the x2 test. P values less than 0.05 were deemed to be statistically significant. 3. Results 3.1. Antimicrobial susceptibility testing Among the 963 E. coli isolates tested, 190 isolates showed resistance to amikacin and 177 of them were positive for 16SRMTase genes. The bulk of the positive isolates possessed rmtB (18.0%) and only 3 (0.3%) and 2 (0.2%) were armA- and rmtEcarrying genes, respectively. Within these 3 groups, only one strain from a 2007 duck isolate harbored both rmtB and armA. The other two armA-positive strains were also from ducks and were isolated in 2007 and 2010. Therefore, only three out of the 10 known 16SRMTase genes were identified in this study. Characterization of the two rmtE-positive strains has been described previously (Xia et al., 2015). Prevalence of amikacin resistance and the rmtB gene among E. coli strains were presented in Fig. 1. The percentages of amikacinresistant isolates were basically consistent with that of rmtBpositive E. coli strains. They presented persistent prevalence from 18.8% and 18.8% in 2002–2005 to 23.3% and 19.8% in 2011–2012, respectively (p > 0.05). All the 173 rmtB-positive isolates were aminoglycosideresistant and meropenem-susceptible. Most strains were resistant to ampicillin, tetracycline, trimethoprim-sulfamethoxazole, nalidixic acid, ciprofloxacin, norfloxacin (>90%) and olaquindox, chloramphenicol, florfenicol and cefotaxime (>65%). The specific information of antimicrobial resistance rates were presented in Fig. S1a (see Supplementary material Fig. S1a in the online version at DOI: 10.1016/j.vetmic.2016.03.018). Moreover, all of the isolates were resistant to three or more classes of antimicrobials (multidrug resistance) tested. Several (28.3%) isolates were resistant to 16 antimicrobial agents, and the most frequently observed pattern of multidrug resistance was AMK-GEN-STR-KANNEO-APR-AMP-CTX-TET-FFC-CHL-SMZ/TMP-NAL-CIP-NOR-OLA. The isolates resistant to 17 or 15 antimicrobial agents were accounted for 21.4% and 17.9%, respectively (Fig. S1b) (see Supplementary material Fig. S1b in the online version at DOI: 10.1016/j.vetmic.2016.03.018). 3.2. PFGE analysis The PFGE results showed diverse genetic profiles among the isolates. The 173 rmtB-positive E. coli isolates were grouped into 112 PFGE clusters apart from the 19 untypeable isolates (data not shown). Although some isolates in one year had identical or similar PFGE patterns, clonal transmission was not detected on the whole. Meanwhile, the 3 armA-positive E. coli isolates showed different PFGE patterns (data not shown). 3.3. 16S-RMTase gene transfer and plasmid analysis In our conjugation experiments, we obtained 116 transconjugants from the 173 rmtB-positive strains with the absence of any armA-positive transconjugants. Transferred antimicrobial-

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resistance phenotypes were aminoglycoside (100%), fosfomycin and cefotaxime (>40%), trimethoprim-sulfamethoxazole, tetracycline, florfenicol, chloramphenicol and olaquindox (>10%). Plasmid replicon typing indicated a wide range of plasmid types. The most prevalent was IncFII (59/116) followed by nontypeable (33/116), IncN (18/116), IncFIA (18/116), IncFIB (22/116), IncI1 (3/116) and IncHI2 (1/116). Further characterization of IncF plasmids indicated that these plasmids consisted of F33 (27/59), F2 (24/59), F43 (5/59) and F34 (3/59). The majority of the IncFIA plasmids belonged to A1 (15/18) and the others were confirmed to be A6 (3/18). IncFIB plasmids were composed of 5 subgroups: B1 (18/22), B12 (1/22), B20 (1/22), B24 (1/22) and B58 (1/22). Specific information on FAB formulas is presented in Table 1. S1-PFGE along with Southern blotting demonstrated that the sizes of rmtB-positive plasmids ranged from 20 kb to 340 kb. However, there were two groups of plasmid sizes consisting of 70–90 kb and 105–120 kb. Among these, 109 transconjugants that carried only one plasmid were used for EcoRI-RFLP analysis. This experiment revealed that a portion of these plasmids belonged to IncF, non-typeable and IncN, isolated in different years or from different origins showed same or similar patterns. Plasmids with sizes of 70, 75, 160 and 105 kb all belonged to the IncF were prevalent in avian and swine isolates from different years (Fig. 2). The non-typeable plasmids with sizes about 75 kb and 85 kb were disseminated in birds. The 70 kb plasmid found both in avian and swine isolates from different years (data not shown). Meanwhile, the plasmid with the size about 60 kb involved in one cluster, both found in birds and swine isolates from different years. It is the most disseminated IncN plasmids (data not shown). 3.4. Detection of other resistance genes Over 90% of the rmtB-positive strains carried one or more other type of resistance gene. The most prevalent genes were oqxAB (61%), floR (47%), blaCTX-M-9G (43%), fosA3 (35%), qepA (32%), aac(60 )-Ib-cr (25%) and blaCTX-1G (14%). qnrB2, qnrS1 and blaCMY-2 were also detected but at a low frequency (<7%). Resistance genotypes transferred to the recipient by conjugation were qepA (55%), blaCTXM-1G (58%) and fosA3 (48%), followed by blaCTX-M-9G (31%) and floR (8%). Among the genes that were co-transferred, blaCTX-M-1G was detected only after 2010 with a low ratio of co-existence (14%) but having a high ratio of co-transfer (58%) (data not shown). The qepA, fosA3 and blaCTX-M-9G genes were continuously detected with rmtB as time passed (Table 2). The co-detection rates of qepA remained high (p > 0.05) but had a significant decrease in co-transfer rate with rmtB (0.01 < P < 0.05). Table 1 Specific information of FAB formulas in rmtB-positive IncF plasmids. FAB formulas

Number of birds

Number of swine

Total number

F33:A-:BF2:A-:BF2:A1:B1 F-:A1:B1 F43:A-:BF34:A-:BF-:A-:B1 F2:A6:B1 F2:A1:B20 F2:A1:B58 F33:A-:B24 F-:A6:B1 F-:A-:B12

15 7 0 1 4 1 2 2 1 1 0 0 1

11 6 7 5 1 2 0 0 0 0 1 1 0

26 13 7 6 5 3 2 2 1 1 1 1 1

Total Number

35

34

69

44

J. Xia et al. / Veterinary Microbiology 188 (2016) 41–46

Fig. 2. EcoRI-RFLP patterns of the four prevalent IncF plasmids. aA, Amikacin; G, Gentamicin; S, Streptomycin; K, Kanamycin; N, Neomycin; R, Apramycin; P, Ampicillin; X, Cefotaxime; E, Cefoxitin; T, Tetracycline; L, Florfenicol; H, Chloramphenicol; S/T, Trimethoprim-Sulfamethoxazole; D, Nalidixic acid; I, Ciprofloxacin; O, Norfloxacin; Q, Olaquindox; F, fosfomycin. Resistance phenotypes transferred to the recipient by conjugation are underlined. bResistance genes transferred to the recipient by conjugation are underlined.

Table 2 Comparisons of co-detection and co-transfer rates of qepA,fosA3 and blaCTX-M-9G with rmtB. Year

2002–2005 (n = 33) 2007–2009 (n = 30) 2010 (n = 64) 2011–2012 (n = 46) p-value

qepA

fosA3

blaCTX-M-9G

Co-detection rate

Co-transfer rate

Co-detection rate

Co-transfer rate

Co-detection rate

Co-transfer rate

21.2% (7/33) 26.7% (8/30) 40.6% (26/64) 32.6% (15/46) p > 0.05

85.7% (6/7) 25.0% (2/8) 69.2% (18/26) 33.3% (5/15) 0.01 < p < 0.05

18.2% (6/33) 30.0% (9/30) 32.8% (21/64) 52.2% (24/46) 0.01 < p < 0.05

16.7% (1/6) 44.4% (4/9) 33.3% (7/21) 70.8% (17/24) 0.01 < p < 0.05

39.4% (13/33) 66.7% (20/30) 46.9% (30/64) 28.3% (13/46) p < 0.01

53.9% (7/13) 50.0% (10/20) 13.3% (4/30) 15.4% (2/13) p < 0.01

The co-detection and co-transfer rates of the fosA3 gene with rmtB both presented significant increases (0.01 < p < 0.05) ranging from 18.2% and 16.7% in 2002–2005 to 52.2% and 70.8% in 2011–2012, respectively. In addition, both co-detection and cotransfer of the blaCTX-M-9G gene showed a significant decrease as time passed (p < 0.01), from 39.4% and 53.9% in 2002–2005 to 28.3% and 15.4% in 2011–2012, respectively (Table 2). 4. Discussion Aminoglycosides continue to play a critical role in therapeutics and are often utilized for their synergistic effects when paired with agents from other classes (Doi et al., 2004). However, the widespread dissemination of 16S-RMTase genes have cast a shadow over aminoglycosides in clinical use. In this study, rmtB was the most prevalent 16S-RMTase gene detected in food-producing animals. This is most likely the result of wide aminoglycoside antibiotic use of agents such as amikacin in food-producing animals. Even more striking was that all the 16SRMTase gene-positive isolates were multidrug–resistant. This

included resistance to b-lactams, fluoroquinolones, tetracyclines, amphenicols and sulfonamides. Actually, almost all 16S-RMTase gene-positive bacterial isolates isolated from human beings or animals have been multidrug resistant. These 16S-RMTase genes often coexist with extended spectrum b-lactamases (ESBLs), PMQR determinants, floR, and fosA3 (Deng et al., 2011b; Yang et al., 2011). In this study, PMQR genes were the most prevalent in 16SRMTase gene-positive isolates, followed by ESBLs, floR and fosA3. Additionally, the blaCTX-M-1G, qepA, fosA3 and blaCTX-M-9G presented relatively high rates of co-transfer during conjugation, even though their prevalence trends differed. Regarding this, we conclude that the prevalence trend of the qepA gene is consistent with that of rmtB, and blaCTX-M-1G and fosA3 are more likely to spread together with rmtB in future. rmtB was detected in 100 isolates (100/479, 20.9%) and 73 isolates (73/484, 15.1%) from birds and swine, respectively. From the years 2002–2012, the positive detection rate of rmtB was stable and averaged about 20%. This indicated that there was a persistent spread of rmtB-positive E. coli strains from 2002 to

J. Xia et al. / Veterinary Microbiology 188 (2016) 41–46

2012 in food-producing animals. Specifically, the prevalence of rmtB was maintained at a relatively high level in both swine and birds (Table S1). This prevalence trend of rmtB should be monitored more closely in the future. rmtB was initially found on a large non-conjugative plasmid in Serratia marcescens from a patient in Japan in 2003 (Doi et al., 2004). It was then reported globally both in human and animals (Wachino and Arakawa, 2012). Among the E. coli strains isolated from humans, the IncF plasmids are most closely related to the dissemination of rmtB. Besides, IncK and IncN have also detected in rmtB-positive E.coli strains (Yu et al., 2010). It has been previously documented that IncF and IncN plasmids contribute to rmtB dissemination among E. coli strains in pig farms (Deng et al., 2011b; Yao et al., 2011). Interestingly, rmtB carried on IncFII plasmids in pets also has a high prevalence (Hou et al., 2012). In this study, IncF (especially IncFII) and non-typeable plasmids played the main role in rmtB dissemination, followed by the IncN plasmids. This is consistent with previous reports indicating that the IncF and IncN plasmids are the primary drivers of dissemination of rmtB in E. coli from swine (Yao et al., 2011). On the other hand, IncF and non-typeable plasmids harboring rmtB were more prevalent in E. coli bird isolates. Since there may be one or more new replicon types in our study group, our future studies will focus on the non-typeable plasmids. Characterization of IncF plasmids indicated that the groups F33 and F2 with the RST formulas A-: B- were the most frequently encountered types in birds and swine. The following prevalent types were F2:A1:B1, F-:A1:B1 and F43:A-:B- in swine and birds, respectively. EcoRI-RFLP results demonstrated a diversity in the clusters among the rmtB-positive plasmids. This diversity of IncF plasmid types has been reported previously (Yang et al., 2015b). However, we still found some plasmids that were responsible for the dissemination of rmtB in food-producing animals. The 75 kb IncF plasmid belonged to F33:A-:B- was disseminated in birds and swine in the years 2005, 2009, 2010, 2011 and 2012. This indicated that there was horizontal transmission among these diverse species in food-producing animals. Another IncF plasmid with a size of 105 kb belonged to F2:A-:B- was disseminated in swine in the years 2004, 2005 and 2007. Among the strains isolated from 2010, the IncF plasmids about 70 kb (F43:A-:B-) and 160 kb (F2:A1: B1) drove the prevalence of rmtB gene in birds and swine, respectively. Both of these two plasmids shared cross-transmission in birds and swine because they were resident in different organs (data not shown). In conclusion, rmtB was the most prevalent 16S rRNA methyltransferase gene and rmtB-positive E. coli strains present persistent spread in food-producing animals in China. IncF plasmids played the major role in rmtB dissemination and the groups F33 and F2 with the RST formulas A-: B- were the most frequently encountered types. These were both prevalent in birds and swine. A group of plasmids belonging to the IncF, non-typeable and IncN groups were also responsible for the dissemination of rmtB among E. coli strains in food-producing animals. Overall, this study suggests that aminoglycoside antibiotics should be used more prudently and the 16S-RMTase distribution in food-producing animals should be monitored.

Acknowledgements This work was supported by the Program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (Grant No. IRT13063), the Special Fund for Agro-scientific Research in the Public Interest (Grant No.

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201203040) and the Natural Science Foundation of Guangdong Province (Grant No. S2012030006590).

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