JAC Advance Access originally published online on March 12, 2007
Journal of Antimicrobial Chemotherapy 2007 59(5):880-885; doi:10.1093/jac/dkm065
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Emergence of RmtB methylase-producing Escherichia coli and Enterobacter cloacae isolates from pigs in China
College of Veterinary Medicine, Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, South China Agricultural University, Guangzhou 510642, People's Republic of China
* Corresponding author. Tel: +86-20-85280237; Fax: +86-20-85284896; E-mail: jhliu{at}scau.edu.cn
Received 27 October 2006; returned 12 November 2006; revised 7 February 2007; accepted 10 February 2007
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Abstract |
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Objectives: To investigate the occurrence of 16S rRNA methylases conferring high-level resistance to aminoglycosides in Enterobacteriaceae isolated from two pig farms in China.
Methods: Enterobacteriaceae isolated from 151 pig rectal swab samples and 9 environmental samples were screened for the presence of the rmtA, rmtB, armA and rmtC genes by PCR and sequencing. Conjugation experiments were carried out to study the transferability of the 16S rRNA methylase genes. All isolates and their transconjugants were tested for susceptibility to antimicrobial agents. The clonal relatedness of RmtB-producing Escherichia coli was assessed by PFGE with XbaI.
Results: Of 152 Enterobacteriaceae isolates recovered from pigs, 49 (32%) were positive for the rmtB gene, including 48 E. coli and a single isolate of Enterobacter cloacae. Of the nine Enterobacteriaceae isolates from environmental samples, no 16S rRNA methylase gene was identified. The 49 rmtB-positive isolates showed resistance to ampicillin, tetracycline and trimethoprim and also carried a blaTEM gene. Transfer of the rmtB and blaTEM genes by conjugation experiments of all 49 isolates was successful, suggesting that the rmtB-containing plasmids in the E. coli and E. cloacae isolates were self-transmissible. Conjugative transfer frequencies varied from 2.2 x 10–10 to 1.3 x 10–6 transconjugants per recipient. The transfer of non-aminoglycoside antimicrobial resistance traits was also observed in most cases. Forty-four rmtB-positive E. coli showed 30 different PFGE types.
Conclusions: The rmtB gene was detected on conjugative plasmids of porcine E. coli and E. cloacae isolates. Both horizontal gene transfer and clonal spread were responsible for the dissemination of the rmtB gene. The emergence of 16S rRNA methylases in Enterobacteriaceae isolates is described for the first time in China. This is also the first report of rmtB-positive Enterobacteriaceae among healthy food-producing animals.
Keywords: 16S rRNA methylases , animal isolates , aminoglycoside resistance
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Introduction |
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Aminoglycosides are important antibiotics in treating severe infections caused by Gram-negative bacteria in clinical settings because of their broad spectrum of target bacteria, potent concentration-dependent bactericidal activity, post-antibiotic effect and ability to act synergistically with many other antibiotics.1 They exert their antibacterial action by binding to the highly conserved A-site of the 16S rRNA of the prokaryotic 30S ribosomal subunits, interfering with protein synthesis and resulting in cell death.2,3 Aminoglycosides are also widely used in food-producing animals to prevent and control bacterial infections and for growth promotion. As a consequence, widespread resistance to different aminoglycosides has emerged. The mechanisms of resistance to aminoglycosides include enzymatic modification of the drug, modification of the ribosomal target and decreased intracellular antibiotic accumulation by alteration of outer membrane permeability, decreased inner membrane transport or active efflux.2–4 Production of aminoglycoside-modifying enzymes is the most common mechanism of resistance to aminoglycosides.
Methylation of 16S rRNA by specific methylases is a mechanism of self-defence of aminoglycoside producers.4 Recently, several 16S rRNA methylases have been identified in clinical isolates of Gram-negative bacteria.5–8 These enzymes conferred very high-level resistance to most clinically important aminoglycosides, including arbekacin, amikacin, kanamycin, tobramycin and gentamicin and could be transferred by conjugation or transformation.5–10 Since the first aminoglycoside resistance methylase named ArmA was reported from France in 2003, a series of 16S rRNA methylases including RmtA, RmtB and RmtC have been found in several countries.5–10 They share 29% to 82% amino acid identity and were detected in several pathogens including Pseudomonas aeruginosa, Serratia marcescens, Escherichia coli and Klebsiella pneumoniae recovered from humans and also one E. coli from the faeces of a diarrhoeic pig in Spain.5–11 RmtA was identified in a clinical strain of P. aeruginosa in Japan and submitted to the DNA Data Bank of Japan in April 2002 (DDBJ accession number AB083212 [GenBank] ).7 The sequence of the armA gene located in Citrobacter freundii isolated in Poland (AF550415 [GenBank] ) was first described in GenBank in October 2002 by Golebiewski et al., though they did not seem to be aware of the function of the gene. In Japan,1,3,4 all of the four genes have been identified, whereas in Taiwan10 and Korea,12 rmtB and armA have been found. In Europe, armA was the most prevalent methylase gene,9,13,14 but Bogaerts et al.15 recently reported for the first time the presence of the rmtB gene in Enterobacteriaceae in Belgium. As the 16S rRNA methylases can confer very high-level resistance to most clinically important aminoglycosides, the dissemination of these genes worldwide has caused a great deal of concern.9,13,16,17
There is clear evidence of adverse human health consequences due to resistant organisms resulting from non-human usage of antimicrobials, so the emergence of bacteria of animal origin, which are resistant to critically important antimicrobial agents for human medicine, such as aminoglycosides, is a great public health problem.11,18 Until now, most 16S rRNA methylase-producing Gram-negative bacteria were recovered from human clinical medicine. Only the armA gene was found in a porcine E. coli isolate from Spain.11 The large amount of various aminoglycosides used in farm animals could have also been a selective pressure for the emergence and spread of pathogenic microbes harbouring the newly identified 16S rRNA methylases.13 In China, several aminoglycosides have been widely used in food-producing animals, including gentamicin, kanamycin, neomycin and amikacin. Aminoglycoside resistance was detected frequently in bacteria from animals in China, with >15% and 50% of E. coli being resistant to amikacin and gentamicin, respectively. This study was conducted to isolate Enterobacteriaceae strains producing 16S rRNA methylases from two pig farms in China and to characterize the isolates with respect to aminoglycoside resistance level, co-resistance to other antimicrobial agents, the transfer of the methylase gene and clonal relationship.
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Sampling and cultivation of bacteria
From October 2005 to February 2006, a total of 160 samples were collected from two swine farms, including 70 samples from Farm A and 90 samples from Farm B. Farm A was located in Guangdong province and Farm B was located in Chongqing, which is 
1000 km away from Guangdong Province. A total of 70 individual pigs from Farm A were randomly selected for rectal swab sampling on the basis of their age and stage of production (sows, finishers, growers and piglets). Of the 90 samples from Farm B, 81 samples were obtained from pigs (rectal swabs), 5 from the surface soil of pigsties, 1 from drinking water and 3 from feed. Samples were seeded in MacConkey agar, and colonies with typical E. coli morphology were selected and subcultured in MacConkey agar for purification. Purified isolates were tested for the presence of 16S rRNA methylase genes. For Farm B, the selected colonies with typical E. coli morphology were subcultured in MacConkey agar containing 128 mg/L gentamicin and 128 mg/L amikacin to screen for gentamicin/amikacin-resistant isolates.
Detection of 16S rRNA methylase genes and the blaTEM gene
The armA, rmtA, rmtB, rmtC and blaTEM genes were detected by PCR. A single colony growing on LB agar was picked and incubated in 2–3 mL of LB broth for 16–18 h. After centrifugation, the bacteria were suspended in 200 µL of sterile double-distilled water and boiled for 10 min and then bathed in ice for 5 min. The supernatant was collected by centrifugation and used for PCR determination. The primers used in this study and the sizes of PCR products are listed in Table 1. PCR reactions were performed in volumes of 50 µL containing 10 x PCR buffer with 1 U of Ex Taq DNA polymerase (TaKaRa), 10 pmol of each primer and 100 µM each dNTP. PCR conditions for the amplification of armA, rmtA, rmtB, rmtC and blaTEM genes were as follows: 5 min at 94°C; 30 cycles of 30 s at 94°C, 30 s at 56°C and 1 min at 72°C and a final extension of 5 min at 72°C. PCR products of the whole coding region of the rmtB gene were cloned using the pMD18-T vector (TaKaRa), transferred into E. coli JM109 and positive clones were selected using an X-Gal/IPTG LB agar plate containing ampicillin (100 mg/L). Recombinant plasmids were purified with QIAGENPrep Plasmid Mini Kit (QIAGEN, Germany) and subjected to DNA sequencing using T7 and SP6 sequence primers on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Tokyo). The obtained DNA sequences were compared with relevant sequences in the GenBank database by using the BLAST algorithm (www.ncbi.nlm.nih.gov). All of the rmtB-positive isolates were identified by classical biochemical methods and were confirmed by using MicrologTM Release 4.2 (Biolog, USA).
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Conjugation experiments
Mobility of 16S rRNA methylase genes was determined by conjugation experiments. E. coli 488 Rifr and streptomycin-resistant E. coli C600 were used as recipients. The donor bacteria and the recipient were grown in tryptic soy broth (TSB) to logarithmic phase, mixed in a 1 : 4 ratio (v/v) and incubated at 37°C for 20 h. Transconjugants were selected on MacConkey agar plates containing amikacin (256 mg/L) and gentamicin (256 mg/L) and rifampicin (256 mg/L) or streptomycin (2048 mg/L). Transfer frequencies were calculated by dividing the number of cfu of transconjugants by the number of cfu of recipients. Antimicrobial susceptibility testing was conducted for transconjugants and the transfer of the rmtB gene was confirmed by PCR as described above.
Antimicrobial susceptibility testing
MICs of ampicillin, ceftiofur, streptomycin, gentamicin, kanamycin, neomycin, amikacin, apramycin, tobramycin, sisomicin, netilmicin, spectinomycin, tetracycline, chloramphenicol, florfenicol, ciprofloxacin and trimethoprim were determined by the broth microdilution method. E. coli ATCC 25922 was used as a quality control strain. Antimicrobial susceptibility testing was conducted and the results were interpreted according to guidelines of the CLSI (formerly the NCCLS).19,20 Extended-spectrum ß-lactamase (ESBL)-producing isolates were screened by the phenotypic confirmatory test recommended by the CLSI.20
PFGE was carried out according to a standard protocol using a CHEF-MAPPER System (Bio-Rad Laboratories, Hercules, CA, USA) in 0.5 x TBE buffer.21 Plugs of genomic DNA were digested with XbaI (TaKaRa). A bacteriophage lambda DNA ladder (Bio-Rad, USA) consisting of 48.5 kb concatemers was used as a size marker. The gels were run at 6.0 V/cm, with an increasing pulse time of 0.5–60 s and an angle of 120° at 14ºC. The running time was 22 h. The results were interpreted according to the criteria published by Tenover et al.22
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Prevalence of methylase genes
A total of 71 isolates were obtained from Farm A, and 23 isolates (22 E. coli and 1 Enterobacter cloacae) were positive for the rmtB gene. As 16S rRNA methylases conferred high-level resistance to both gentamicin and amikacin, only those isolates from Farm B that grew on MacConkey agar supplemented with 128 mg/L gentamicin and 128 mg/L amikacin were selected for PCR amplification. A total of 26 amikacin/gentamicin-resistant E. coli isolates from pigs were obtained, and all were positive for the rmtB gene. Of the nine Entero- bacteriaceae isolates from environmental samples, none of the 16S rRNA methylase genes was identified. Thus, a total of 49 (32%) of the 152 Enterobacteriaceae isolates recovered from pig rectal swab samples were positive for the rmtB gene. The 49 rmtB-positive isolates also contained the blaTEM gene. Sequence analysis of the rmtB amplicons showed that they did not differ from the published sequence (AB103506 [GenBank] ). The sequences have been submitted to GenBank (accession numbers DQ355981 [GenBank] and EF017943 [GenBank] ). The rmtB-positive E. cloacae was recovered from a pig that also harboured an rmtB-positive E. coli, suggesting that in vivo horizontal transfer of the resistance determinant had possibly occurred.
RmtB was first identified in S. marcescens from Japan in 2004 and subsequently was found in K. pneumoniae and E. coli isolates from Taiwan, Korea and Belgium.7,10,12,15 To date, the prevalence of studied bacteria carrying 16S rRNA methylase genes appears to be low (<1.0% in 4100 combined test isolates from Taiwan10 and 0.15% in 15 386 Enterobacteriaceae clinical isolates from two Belgian hospitals15), and the isolates were mostly from human clinical medicine. But in this study, to our surprise, an unusually high prevalence of rmtB was detected among Enterobacteriaceae isolates from two pig farms. Of great concern is that all the rmtB-positive isolates were recovered from the commensal flora of healthy pigs. The commensal bacterium constitutes a reservoir of resistance genes for (potentially) pathogenic bacteria and plays an important role in the ecology of antimicrobial resistance of bacterial populations.23 Thus, dissemination of 16S rRNA methylases to pathogenic bacteria from commensal Enterobacteriaceae of food-producing animals should not be ruled out.
The reason for the high prevalence of rmtB in isolates of porcine origin in this study and the origin of the 16S rRNA methylase genes are still unknown. Galimand et al.6 speculated that armA could have originated from as yet unknown aminoglycoside-producing actinomycetes. Some authors indicated that Gram-negative bacterial species from animals and the environment may be reservoirs for emerging antibiotic resistance genes spreading in human pathogens.24 We speculate that aminoglycosides used in animals may play an important role in the emergence and spread of 16S rRNA methylase genes.
No other kind of the 16S rRNA methylase gene was detected except rmtB. To date, the rmtA gene has only been found in P. aeruginosa, and rmtC has only been detected in Proteus mirabilis.5,8 But armA and rmtB are widely distributed in most Enterobacteriaceae.6,7,10,13 In the present study, the rmtB gene was detected for the first time in E. cloacae.
Antimicrobial susceptibility analysis
Table 2 shows the MIC ranges, MIC50s and MIC90s of 10 aminoglycosides and 7 non-aminoglycoside antimicrobial agents for the 49 rmtB-positive isolates. They all showed extraordinarily high-level resistance to 4,6-substituted deoxystreptamine aminoglycosides, including gentamicin, kanamycin, tobramycin, amikacin, netilmicin and sisomicin (MIC90s > 1024 mg/L), but not to 4,5-substituted deoxystreptamine antimicrobials, such as neomycin, and other aminoglycosides and aminocyclitol antibiotics including streptomycin, apramycin and spectinomycin as reported in another study.7 The 49 rmtB-containing strains also showed resistance to more than two non-aminoglycoside agents commonly used in animal farms. Resistance rates of these isolates to ampicillin, tetracycline, trimethoprim, ciprofloxacin and chloramphenicol were 100%, 100%, 100%, 59.2% and 71.4%, respectively. The percentages of isolates that have MICs of florfenicol 
32 mg/L, streptomycin 64 mg/L, spectinomycin 128 mg/L, neomycin 16 mg/L and apramycin 32 mg/L were 42.9%, 91.8%, 87.7%, 75.5% and 26.5%, respectively. But unlike the results of other studies that found armA and rmtB are always associated with CTX-M-type ESBL genes,10,15 rmtB-positive isolates of this study showed susceptibility (MIC <8 mg/L) to ceftiofur (a third-generation cephalosporin) and were negative in the phenotypic ESBL confirmatory test. Our previous studies showed that there was only a low detection of ESBLs in E. coli isolates from food-producing animals in China, though the dissemination of ESBLs among human clinical isolates is very serious.25 These results may be due to the less frequent use and later use of extended-spectrum cephalosporins in food animals compared with human clinical medicine in China, as ceftiofur is the only broad-spectrum cephalosporin approved for systemic use in food-producing animals since 2002 in China (www.agri.gov.cn/blgg/t20021219_36976.htm).
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Transferability of rmtB gene by conjugation
Amikacin/gentamicin-resistant transconjugants were successfully obtained from all 49 RmtB-producing isolates by conjugation. Conjugative transfer frequencies varied from 2.2 x 10 – 10 to 1.3 x 10 – 6 transconjugants per recipient. All transconjugants showed very high-level resistance to all 4,6-substituted deoxystreptamine aminoglycosides, like their parental strains (Table 3). The presence of the rmtB gene was confirmed in all transconjugants by PCR analysis.
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The transfer of 4,5-substituted deoxystreptamine antimicrobial and non-aminoglycoside antimicrobial resistance traits was also observed in some isolates (Table 3), suggesting that self-transmissible resistance genes other than rmtB exist in these isolates. Two transconjugants with different resistance patterns from one parental strain were also obtained, indicating that the rmtB gene could be located in different self-transmissible plasmids or the loss of some of the resistance determinants might have occurred during conjugation.
All 52 transconjugants were resistant to ampicillin and trimethoprim, and >50% of them were also resistant to tetracycline and chloramphenicol. Fifty transconjugants showed 25 different resistance patterns for four 4,5-substituted deoxystreptamine antimicrobials and seven non-aminoglycoside antimicrobials (Table 3). Since we were not able to obtain restriction fragment patterns of the rmtB-carrying conjugative plasmids, statements on the structural differences or similarities of these plasmids are not possible. However, the variable resistance phenotypes expressed by the transconjugants indicate that the rmtB gene might have been exchanged between different conjugative plasmids.
In Japan, the right end of transposon Tn3 including blaTEM-1 was found to be upstream of rmtB, and three rmtB-positive transconjugants of Taiwan all expressed TEM-1.7,10 Fifty transconjugants obtained in this study all showed resistance to ampicillin and harboured a blaTEM gene. These findings suggest that the genetic environment of rmtB in the 48 E. coli and 1 E. cloacae might be similar to that found in Japan and Taiwan and that RmtB-producing Gram-negative microbes that harbour a very similar genetic environment carrying the rmtB gene might have spread across Asia.
Clonal relationship of RmtB-producing isolates by PFGE
Of the 48 rmtB-positive E. coli isolates, 44 were successfully typed by PFGE, and a total of 30 different PFGE profiles were obtained (Table 3). Conjugation experiments showed that rmtB-containing plasmids transferred easily to recipients. These findings suggested that the transfer of the rmtB gene was not solely due to the spread of a specific rmtB-positive E. coli clone, but both horizontal gene transfer and clonal spread were responsible for the dissemination of the rmtB gene in bacteria of animal origin in China.
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Conclusions |
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In conclusion, we have described for the first time the occurrence of plasmid-mediated 16S rRNA methylases conferring high-level aminoglycoside resistance to Enterobacteriaceae in China, and this is the first report of the emergence of rmtB-positive Enterobacteriaceae among commensal flora of healthy food-producing animals. Both horizontal gene transfer and clonal spread were responsible for the dissemination of the rmtB gene. The emergence of high-level aminoglycoside resistance conferred by 16S rRNA methylases in Enterobacteriaceae from food-producing animals is a serious clinical problem, as the resistance determinants may be transmitted to humans via the food chain. Thus, it is necessary to monitor the spread of such resistant bacteria in both humans and animals.
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None to declare.
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Acknowledgements |
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We are very grateful to Dr Sheng Chen (Department of Microbiology and Molecular Genetics, Medical College of Wisconsin), Professor Ming-Gui Wang (Division of Infectious Diseases, Huashan Hospital, Fudan University) and Professor Xing-Quan Zhu for improving this manuscript. This work was supported in part by research grants from the National Natural Science Foundation of China (30130140 and U0631006).
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References |
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