First report of the IncI1/ST898 conjugative plasmid encoding rmte2. 16S rrna methyltransferase gene in Escherichia coli

Transcription

1 AAC Accepted Manuscript Posted Online 21 September 2015 Antimicrob. Agents Chemother. doi: /aac Copyright 2015, American Society for Microbiology. All Rights Reserved. [First Authors Last Name] Page First report of the IncI1/ST898 conjugative plasmid encoding rmte2 16S rrna methyltransferase gene in Escherichia coli Jing Xia a, Jian Sun a, Liang Li a, Liang-Xing Fang a, Hui Deng a, Run-Shi Yang a, Xing-Ping Li a, Xiao-Ping Liao a a, b#, Ya-Hong Liu a Laboratory of Veterinary Pharmacology, College of Veterinary Medicine, South China Agricultural University, Guangzhou , P. R. China, b Jiangsu Co-Innovation Centre for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, the People s Republic of China # Corresponding author: Prof. Ya-Hong Liu, College of Veterinary Medicine, South China Agricultural University, Guangzhou , PRC Tel: ; Fax: ; gale@scau.edu.cn Manuscript Type: Letter to the editor Running title: rmte2 in E. coli Keywords: Escherichia coli, rmte2, 16S rrna methyltransferase, IncI1, ST898 [Insert Running title of <72 characters]

2 Aminoglycoside antibiotics such as amikacin and gentamicin, often along with β-lactams, have been used for the treatment of serious infections caused by Gram-negative and Grampositive pathogenic bacteria in clinical settings (1). However, bacteria have been furnished with various resistance mechanisms to become resistant to aminoglycosides (2). Recently, the spread of 16S rrna methyltransferase (16S-RMTase), responsible for very-high-level resistance against various aminoglycosides (MIC >256 μg/ml), has become apparent in both human and veterinary medicine (1). From the first report in 2003, ten 16S-RMTase encoding genes, named rmta, rmtb, rmtc, rmtd, rmte, rmtf, rmtg, rmth, arma and npma, were identified (3). Of the known 16S RMTases, rmte has been reported in only two Escherichia coli strains, which were identified from a calf and a patient in the United States, respectively (4, 5). Here, we reported the first rmte-producing E. coli isolated from swine in China. A total of 963 non-duplicate E. coli isolates (479 from avian and 484 from swine), isolated from diseased food-producing animals between 2002 and 2012 in Guangdong, China, were used in this research. Among them, 277 swine isolates and 183 avian strains were derived from farms, while the other isolates were recovered from diagnostic labs. E. coli S68 and E. coli 87 were isolated from diseased pigs in 2005 and 2010, respectively. The two strains were isolated in two farms, but located in one district. Both of them exhibited high-level resistance to amikacin (>256 μg/ml) and gentamicin (>256 μg/ml). PCR detection and sequencing of 16S-RMTase encoding genes then indicated that rmte was present in both strains. However, a single base mutation of T C was detected at nucleotide 20 of rmte, which causes a substitution of Val (7) by Ala in the gene product. According to the nomenclature rules suggested, we would like to designate it as rmte2 (6).

3 PFGE with XbaI was performed as described previously (7). Two isolates shared identical PFGE types (data not shown), which suggested the spread of rmte2 was probably due to a specific E. coli clone. Phylogenetic typing and multilocus sequence typing (MLST) were conducted as described previously (8, 9). The results assigned the two strains to phylogenetic group A and sequence type 898 (ST898), different from the rmte-positive E. coli isolate YDC637 of human origin, which is phylogenetic group D and sequence type 354 (ST354) (5). Conjugation experiments by the broth-mating method were conducted as previously described (10). E. coli C600, highly resistant to streptomycin, was used as the recipient in the conjugation experiments. Two transconjugants were successfully obtained, with high-level resistance to amikacin and gentamicin (Table 1). The two plasmids showed identical EcoRI restriction digestion profiles (Figure 1b). And the size of the plasmids carrying rmte2 were estimated to be approximately 110 kb by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and southern-blotting (11) (Figure 1a), and its replicon type (PBRT) was determined to be IncI1 (12), while the rmte-carrying pydc637 was characterized to be an IncA/C plasmid (13). This plasmid conferred resistance only to aminoglycosides, but susceptible to other classes of agents (Table1). PCR mapping according to the sequences of the plasmid pydc637 suggested that ISCR20-like transposase was located upstream of rmte2, but harbored a 697-bp region in the interval. Sequences downstream of rmte2 could not be determined due to lack of amplification. We then confirmed it by RETS-PCR (14), and the result indicated ISVs1-like transposase was located downstream rmte2 (Genbank accession number: KT428293). ST898 were only reported in two pigs and one dog in Germany ( And according to recent reports, IncI1 plasmids, ranged from 80 kb to 130 kb, appears to have a close association with extended-

4 spectrum β-lactamase, especially with CTX-Ms (15). Among the 16S rrna methyltransferase genes, rmtb and arma are commonly identified in E. coli, while rmte is rarely detected, therefore the routine surveillance of rmte in aminoglycoside-resistant Enterobacteriaceae should be warranted. When the MICs of amikacin and gentamicin were found above 256 μg/ml, both rmtb, arma and other rarely seen16s rrna methyltransferase genes (like rmte) should be detected. In the other hand, the amikacin was widely used in food-producing animals in China. Based on the differences in the sequences, origin of the strains, STs, plasmid incompatibility groups and genetic environment of the genes, rmte2 may have been acquired by E. coli in China in an event separate from rmte1 in the United States. In conclusion, here we reported two rmte2-positive E. coli strains of swine origin belonged to phylogenetic group A and ST898. Of note, it may be speculated that the clone and plasmid carrying the rmte2 gene could spread by both vertical and horizontal transfer. To our knowledge, this is the first report of rmte2 in China. The further characterization of the IncI1 plasmid is to be done. 76

5 Acknowledgements This work was supported by the National Science Fund for Distinguished Young Scholars (Grant No ), the Special Fund for Agro-scientific Research in the Public Interest (Grant No ) and the Natural Science Foundation of Guangdong Province (Grant No. S ) References 1. Wachino J, Arakawa Y Exogenously acquired 16S rrna methyltransferases found in aminoglycoside-resistant pathogenic Gram-negative bacteria: an update. Drug Resist Updat 15: Poole K Aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 49: O'Hara JA, McGann P, Snesrud EC, Clifford RJ, Waterman PE, Lesho EP, Doi Y Novel 16S rrna methyltransferase RmtH produced by Klebsiella pneumoniae associated with war-related trauma. Antimicrob Agents Chemother 57: Davis MA, Baker KN, Orfe LH, Shah DH, Besser TE, Call DR Discovery of a gene conferring multiple-aminoglycoside resistance in Escherichia coli. Antimicrob Agents Chemother 54: Lee CS, Hu F, Rivera JI, Doi Y Escherichia coli sequence type 354 coproducing CMY-2 cephalosporinase and RmtE 16S rrna methyltransferase. Antimicrob Agents Chemother 58:

6 Doi Y, Wachino J, Arakawa Y Nomenclature of plasmid-mediated 16S rrna methylases responsible for panaminoglycoside resistance. Antimicrob Agents Chemother 52: Gautom RK Rapid Pulsed-Field Gel Electrophoresis Protocol for Typing of Escherichia coli O157:H7 and Other Gram-Negative Organisms in 1 Day. J Clinical Microbiology 35: Clermont O, Bonacorsi S, Bingen E Rapid and Simple Determination of the Escherichia coli Phylogenetic Group. Applied And Environmental Microbiology 66: Wirth T, Falush D, Lan R, Colles F, Mensa P, Wieler LH, Karch H, Reeves PR, Maiden MC, Ochman H, Achtman M Sex and virulence in Escherichia coli: an evolutionary perspective. Mol Microbiol 60: Chen L, Chen ZL, Liu JH, Zeng ZL, Ma JY, Jiang HX Emergence of RmtB methylase-producing Escherichia coli and Enterobacter cloacae isolates from pigs in China. J Antimicrob Chemother 59: Barton BM, Harding GP, Zuccarelli AJ A General Method for Detecting and Sizing Large Plasmids. Analytical Biochemistry 226: Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 63: Lee C-S, Li J-J, Doi Y Complete Sequence of Conjugative IncA/C Plasmid Encoding CMY-2 β-lactamase and RmtE 16S Ribosomal RNA Methyltransferase. Antimicrobial Agents and Chemotherapy doi: /aac :aac

7 Kusumoto M, Fukamizu D, Ogura Y, Yoshida E, Yamamoto M, Iwata T, Ooka T, Akiba M, Hayashi T Lineage-Specific Distribution of Insertion Sequence Excision Enhancer in Enterotoxigenic Escherichia coli Isolated from Swine. Applied and Environmental Microbiology 80: Yang X, Liu W, Liu Y, Wang J, Lv L, Chen X, He D, Yang T, Hou J, Tan Y, Xing L, Zeng Z, Liu JH F33: A-: B-, IncHI2/ST3, and IncI1/ST71 plasmids drive the dissemination of fosa3 and bla CTX-M-55/-14/-65 in Escherichia coli from chickens in China. Front Microbiol 5:

8 131 Table 1 Characterization of the two isolates carrying rmte2 Phyloge MIC (μg/ml) Strains Origin Year Resistant spectrum Resistant genes netic PBRT MLST AMK GEN KAN group S68 Swine 2005 AMP/TET/OQX/CHL/FFC/NAL/NOR/CIP/ SMZ/TMP/ NEO/AMK/STR/KAN/GEN rmte2, bla TEM-1, oqxab, flor A >256 >256 >256 FII, I1 ST898 JS68 AMK/STR/KAN/GEN rmte2, bla TEM-1 >256 >256 >256 I1 87 Swine 2010 AMP/TET/OQX/CHL/FFC/NAL/NOR/CIP/ SMZ/TMP NEO/AMK/STR/KAN/GEN rmte2, bla TEM-1, oqxab, flor A >256 >256 >256 FII, I1 ST898 J87 AMK/STR/KAN/GEN rmte2, bla TEM-1 >256 >256 >256 I1 132 J, corresponding transconjugants; AMP, Ampicillin; TET, Tetracycline; OQX, Olaquindox; CHL, Chloramphenicol; FFC, Florfenicol; NAL, Nalidixic acid; NOR, Norfloxacin; 133 CIP, Ciprofloxacin; SMZ/TMP, Trimethoprim-Sulfamethoxazole; NEO, Neomycin; AMK, Amikacin; STR, Streptomycin; KAN, Kanamycin; GEN, Gentamicin

9 Figure 1 (a) Plasmids profile of the two rmte2 positive strains. Lane M: Low Ranger PFG Marker; Lanes 1 and 3: strain S68; Lanes 2 and 4: strain 87. (b) EcoRI restriction digestion profiles of rmte2-carrying plasmids of the two transconjugants. Lane M: λhindiii marker; Lanes 1 and 3: strain JS68; Lanes 2 and 4: strain J

10 Figure 2 Genetic environments of the two E. coli carrying rmte2