Emergence of ArmA and RmtB aminoglycoside resistance 16S rrna methylases in Belgium

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1 Journal of Antimicrobial Chemotherapy (2007) 59, doi: /jac/dkl527 Advance Access publication 15 January 2007 Emergence of ArmA and RmtB aminoglycoside resistance 16S rrna methylases in Belgium Pierre Bogaerts 1 *, Marc Galimand 2, Caroline Bauraing 1, Ariane Deplano 3, Raymond Vanhoof 4, Ricardo De Mendonca 3, Hector Rodriguez-Villalobos 3, Marc Struelens 3 and Youri Glupczynski 1 1 Laboratoire de Bactériologie, Cliniques Universitaires UCL de Mont-Godinne, 1 Av. Dr Gaston Thérasse, B-5530 Yvoir, Belgium; 2 Unité des Agents Antibactériens, Institut Pasteur, Paris Cedex, rance; 3 Laboratoire de Bactériologie, Hôpital Universitaire Erasme ULB, B-1070 Brussels, Belgium; 4 Unité des Antibiotiques, Institut Pasteur de Bruxelles, B-1180 Brussels, Belgium Received 15 September 2006; returned 24 November 2006; revised 29 November 2006; accepted 4 December 2006 Objectives: 16S rrna methylase-mediated high-level resistance to aminoglycosides has been reported recently in clinical isolates of Gram-negative bacilli only from a limited number of countries. This study was conducted to investigate the occurrence of this type of resistance in clinical isolates of Enterobacteriaceae from two Belgian hospitals and the characteristics of the strains. Methods: We screened for high-level gentamicin, tobramycin and amikacin resistance in clinical isolates of Enterobacteriaceae consecutively collected between 2000 and 2005 at two laboratories by PCR for the arma, rmta and rmtb 16S rrna methylase genes. The b-lactamase presence in the strains was also determined by phenotypic and genotypic methods. Results: Overall arma genes were detected in 18, Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae and Citrobacter amalonaticus whereas rmtb was detected in a single E. coli isolate. The rmta gene was not found. All 16S rrna methylase-bearing strains produced extendedspectrum b-lactamases (ESBLs), predominantly type CTX-M-3, as well as various types of b-lactamases. In the majority of the strains, the arma gene was carried by conjugative plasmids of the IncL/M incompatibility group whereas rmtb was borne by an IncI plasmid. Conclusions: This is the first report of the emergence of 16S rrna methylases in Enterobacteriaceae in Belgium. The rapid spread of multidrug-resistant isolates producing both ESBLs and 16S rrna methylases raises clinical concern and may become a major therapeutic threat in the future. Keywords: Enterobacteriaceae, resistance genes, extended-spectrum b-lactamases Introduction Aminoglycoside antibiotics are widely used in the treatment of a broad range of life-threatening infections caused by Gram-negative bacteria. 1 They bind to the highly conserved A site of the 16S rrna of the bacterial 30S ribosomal subunit, interfering with protein synthesis with subsequent bacterial death. 2 4 Resistance to aminoglycosides is primarily due to enzymatic modification of the drug through N-acetylation, O-nucleotidylation or O-phosphorylation. 5,6 Other less common mechanisms of resistance include decreased intracellular uptake of the antibiotic by altering the outer membrane permeability, less inner membrane transport or active efflux, or modification of the target by mutation in ribosomal proteins or in 16S rrna. 2,4 Plasmid-encoded 16S rrna methylases have recently emerged as a new mechanism of resistance to aminoglycosides in Enterobacteriaceae and in nonfermentative Gram-negative bacilli The first reported 16S rrna methylase gene arma was found on a plasmid of a clinical isolate from rance. 7 This enzyme belongs to the m 7 G-methyltransferase class of anti-suicide methylases, which are naturally found in aminoglycoside-producing organisms such as Streptomyces spp. and Micromonas spp. Unlike modifying enzymes that vary in their substrate ranges, acquired methylases confer high-level resistance to all clinically important aminoglycosides except streptomycin, by post-transcriptional methylation of the G1405 residue of 16S rrna. 12 This leads to loss of affinity... *Corresponding author. Tel: þ ; ax: þ ; pierre.bogaerts@mont.ucl.ac.be # The Author Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. or Permissions, please journals.permissions@oxfordjournals.org

2 Bogaerts et al. for aminoglycosides. The structural genes for the methylases are often located on self-transferable plasmids and are frequently associated with genes for extended-spectrum b-lactamases (ESBLs), mostly bla CTX-M. 13 Sequencing of the genetic environment of arma revealed that this gene is located on a transposon offering a supplemental way of dissemination of resistance. 7,13 Dissemination of arma to various species of Enterobacteriaceae has been reported in several European countries 13 as well as in South-East Asia 8,14,15 and this gene has also been detected in an Escherichia coli isolate from veterinary source. 16 Other novel 16S rrna methylase genes, rmta, rmtb and rmtc have been detected recently in Pseudomonas aeruginosa, 10,14 Serratia marcescens 9 and Proteus mirabilis, 11 and the spread of such resistance determinants has become a great concern. The occurrence of high-level aminoglycoside resistance mediated by 16S rrna methylases among clinical isolates of Gram-negative bacilli in Belgium has not been previously assessed. The aim of this study was to investigate the occurrence of 16S rrna methylases in aminoglycoside-resistant isolates from two hospitals in Belgium and to characterize the host bacteria. Materials and methods Clinical isolates We screened by disc diffusion non-duplicate Enterobacteriaceae collected between January 2000 and December 2005 at two Belgian hospitals (Erasme hospital, a 900-bed university hospital in Brussels, and Mont-Godinne university hospital (MGUH), a 380-bed hospital in the province of Namur). All species were identified by conventional methods 17 or by using GN cards on the VITEK w 2 system (biomérieux, Marcy l Étoile, rance). The selection criterion was concomitant resistance to gentamicin, tobramycin and amikacin. Antimicrobial susceptibility testing MICs of antimicrobials were determined by microbroth dilution using a Cooke Dynatech MIC2000 inoculator (Alexandria, VA, Table 1. Primers used for PCR detection and sequencing of resistance genes USA) or by the VITEK w 2 system according to CLSI guidelines and interpretative criteria. 18 E. coli ATCC and P. aeruginosa ATCC were used as reference strains. Apramycin, neomycin and streptomycin were tested by disc diffusion using NeoSensitabs tablets (Rosco w, Taastrup, Denmark) and the results were interpreted according to the recommendations of the manufacturer. Detection of 16S rrna methylase genes The arma, rmta and rmtb genes were detected by PCR. A fresh bacterial colony was suspended in 100 ml of sterile distilled water and boiled at 1008C for 10 min. After centrifugation, 3 ml of supernatant was used for PCR assays with the primers described in Table 1. Amplification of DNA was performed in a 9700 thermal cycler (Applied Biosystems, oster City, CA, USA). PCR elongation times and temperature conditions were as follows: 948C for 5 min followed by 30 cycles of 948C for 30 s, 558C for 1 min and 728C for 1 min; and a final extension at 728C for 7 min. PCR products were electrophoresed in 1.5% agarose gels and visualized under UV light. PCR products were then purified with the QIA quick PCR purification kit (QIAGEN, Inc., Chatsworth, CA, USA) and sequenced on both strands with an ABI PRISM 3100 Genetic analyser (Applied Biosystems). The sequences were compared with those in GenBank nucleotide database at Detection of aminoglycoside resistance genes Genes aac(6 0 )-Ia, aac(6 0 )-Ib, aac(6 0 )-Ic, aac(6 0 )-Id, aac(6 0 )-If, aac(6 0 )-Ig, aac(6 0 )-Ih, aac(6 0 )-Ij, aac(6 0 )-Ik, aac(6 0 )-Il, aac(6 0 )-Im, aac(6 0 )-IIa, aac(6 0 )-IIb, aac(3)-ia, aac(3)-iic, aac(3)-iiia, aac(3)-iia, aac(2 0 )-Ia and ant(2 00 )-Ia encoding aminoglycoside modifying enzymes were detected using PCR as previously described. 19,20 b-lactamase characterization ESBL production was detected by double disc synergy test using cefotaxime, ceftazidime, cefepime and amoxicillin/clavulanate Primer Target Sequence Amplicon size (bp) Reference met arma caa atg gat aag aat gat gtt metr tta ttt ctg aaa tcc act rmtb W rmtb atg aac atc aac gat gcc ct rmtb RV cct tct gat tgg ctt atc ca rmta W rmta cta gcg tcc atc ctt tcc tc rmta RV ttt gct tcc atg ccc ttg cc TEM W1 bla TEM caa ctc ggt cgc cgc ata cac ta 502 this work TEM RV1 cgg gag ggc tta cca tct ggc SHV 65W bla SHV cga aag atc cac tat cgc cag cag 234 this work SHV 65RV att cag ttc cgt ttc cca gcg g CTXG1 W 1 bla CTX group 1 agt tca cgc tga tgg cga cg 676 this work CTXG1 RV 3 aac cca gga agc agg cag tcc CTXG2 W1 bla CTX group 2 atg atg act cag agc att cgc cg 827 this work CTXG2 RV 1 cgg ctt tcc gcc ttc tgc tc CTXG9 W1 bla CTX group 9 gcc gaa aaa cac gtc aac ggc 472 this work CTXG9 RV1 gaa cca gcg gcg cac gac 460

3 Aminoglycoside 16S rrna methylases in Belgium discs and by the confirmatory double disc combination test using ceftazidime (30 mg) and cefotaxime (30 mg) discs alone and in association with clavulanic acid (10 mg) as recommended by the CLSI. 18 Crude b-lactamase extracts were prepared from overnight growth on nutrient agar slopes. Bacteria were washed into 2 ml amounts of 0.01 M phosphate buffer, ph 7.0 chilled on ice and sonicated for 3 20 s. Isoelectric focusing (IE) was carried out by the method of Matthew et al. 21 with a Phastsystem (Amersham Biosciences, Uppsala, Sweden) on ph 3 9 PhastGel w according to the manufacturer s protocol except that migration was performed at 500 V instead of 2000 V. b-lactamase activity was detected by overlaying the gel with 100 ml of a 500 mg/l nitrocefin solution (Oxoid, Basingstoke, UK). PCR detection of bla TEM /bla SHV /bla CTX-M -related genes was carried out with the oligonucleotide primers described in Table 1 using the same PCR conditions as those described for detection of 16S rrna methylase genes except that the annealing step was performed at 628C. The sequences of the CTX-M-1 and CTX-M-9 group amplicons were obtained with primers described previously. 22,23 The sequences were compared with those in the GenBank nucleotide database at PGE analysis Macrorestriction (XbaI) analysis resolved by PGE was performed as previously described with PGE separation conditions of s for 24 h. 24 PGE patterns were analysed with the Dice coefficient and the unweighted pair group method with average linkages (UPGMA) clustering method using BioNumerics software (Applied Maths, Kortrijk, Belgium). The PGE classification criteria were described previously and include a type, designated by a capital letter (e.g. A) and patterns showing 0 6 DNA fragment differences. 25 Conjugation experiments Conjugation was performed on solid or in liquid medium with nalidixic acid-resistant E. coli BM694 or rifampicin-resistant E. coli C600 or streptomycin-resistant E. coli HB101 as recipients depending on the resistance phenotype of the donor strain. Selection of the transconjugants was performed on BHI agar supplemented with rifampicin (250 mg/l), streptomycin (500 mg/l) or nalidixic acid (300 mg/l) and amikacin (250 mg/ L). In each mating experiment, randomly selected transconjugants were purified and tested for antibiotic resistance. Transconjugants were analysed by PCR and their resistance profiles were determined as described above. Identification and typing of incompatibility groups of plasmids were performed on all transconjugants by PCR (inc/rep PCR) of the major plasmid incompatibility groups among Enterobacteriaceae as described by Carattoli et al. 26 Results and discussion Twenty-two isolates were resistant to gentamicin, tobramycin and amikacin by disc diffusion testing. Of these, 18 [K. (10), E. coli (4), Enterobacter aerogenes (2), Enterobacter cloacae (1), Citrobacter amalonaticus (1)] were resistant to high levels (MIC.512 mg/l) of kanamycin, gentamicin, tobramycin, netilmicin and amikacin (Table 2). The arma gene was detected in 18 isolates including one E. coli strain (#4470) with a lower level of resistance to amikacin (MIC of 8 mg/l) whereas rmtb was detected in a single E. coli strain (#1540). No resistant strains were found to be positive for the rmta gene. The arma and rmtb amplicons were sequenced and showed 100% identity with the arma gene in K. BM and rmtb in S. marcescens S The aac(6 0 )-Ib gene (encoding an aminoglycoside modifying enzyme) was detected in association with either ant(2 00 )-Ia or aac(3)-iic in three arma- and rmtb-negative aminoglycoside-resistant strains with lower MICs of amikacin (MICs of 8, 16 and 32 mg/l, respectively) (not shown). An aac(3)-iic gene was detected in 17 16S rrna methylaseproducing isolates. In two K. isolates (#2432 and #2451) aac(3)-iic and aac(6 0 )-Ia were detected in association with arma. No genes encoding aminoglycoside modifying enzymes were found in the rmtb-positive strain. All 19 isolates were found to be susceptible to apramycin (inhibition zones ranging between 23 and 27 mm) and all but two (C. amalonaticus #3562 and E. coli #2312, inhibition zones of 11 and 14 mm, respectively) were also susceptible to neomycin (inhibition zones were between 21 and 30 mm). Overall, the aminoglycoside resistance profiles of the selected strains encompassing all 4,6-disubstituted deoxystreptamines (kanamycin, gentamicin, tobramycin, netilmicin and tobramycin) were consistent with a resistance mechanism due to 16S rrna methylases though resistance to neomycin (strains #3562 and #2312) and streptomycin suggested the presence of additional aminoglycoside resistance mechanisms since 16S rrna methylase genes do not confer resistance to these antibiotics. 7 All but one 16S rrna methylase-producing Enterobacteriaceae were collected from specimens originating from patients hospitalized at Erasme University Hospital between October 2000 and June The rmtb-positive strain was collected from MGUH and was isolated in November 2004 from a urinary specimen of a Serbian patient transferred from Belgrade s hospital after stroke. The sites of infection or colonization by the arma-positive isolates recovered at Erasme hospital from the 15 patients were as follows: respiratory in 7 patients; gastrointestinal in 5 patients; urinary in 2 patients; and blood, in 1 patient. Of the 15 patients, 12 stayed at the ICU when the arma-positive isolates were recovered while the other three each stayed at different wards. Overall, 10 patients acquired infection 3 days or more after admission and five were considered colonized or infected prior to admission. our of them were Algerian citizens with complicated medical pathologies who had been hospitalized at different hospitals in Algeria and had been transferred to Belgium for diagnostic work-up and medical treatment. The index case patient was a 49-year-old man with a complicated head trauma and pneumonia who was admitted in October Thereafter clustered secondary cases of infections with arma-positive K. strains occurred in December 2000 (two patients) and in May 2001 (five patients) among patients hospitalized in the same ICU. b-lactamase characterization All 19 arma- and rmtb-positive Enterobacteriaceae isolates were found to co-produce an ESBL. The b-lactamase genes detected by PCR and identified by DNA sequencing are summarized in Table 2. The pis of the b-lactamases determined on IE gels, 461

4 Table 2. PGE, methylase and CTX-M gene identification and MIC values for clinical isolates and transconjugants Isolate No. PGE Clinical isolates 16S rrna methylase gene bla CTX-M AMK a CAZ CTX EP MER CIP SXT Transconjugants 16S rrna methylase gene bla CTX-M AMK a CAZ CTX EP MER CIP SXT 462 Citrobacter 3562 NA arma CTX-M ,0.25, arma CTX-M ,0.25,0.25,20 amalonaticus Enterobacter 3511 A arma CTX-M ,0.25, arma CTX-M-3 512,1 4,1,0.25, aerogenes Enterobacter 5064 B arma CTX-M ,0.25, arma CTX-M ,0.25,0.25,20 aerogenes Enterobacter 3544 NA arma CTX-M ,0.25, arma CTX-M-3 512,1 4,1,0.25, cloacae Escherichia coli 1540 C rmtb CTX-M ,1 32 2, rmtb none 512,1,1,1, Escherichia coli 2312 D arma CTX-M-3 512,1 8 2,0.25, arma CTX-M ,0.25,0.25,20 Escherichia coli 4461 E arma CTX-M-3 512,1 4 2,0.25, arma CTX-M-3 512,1 4,1,0.25, Escherichia coli 4470 H arma CTX-M , arma CTX-M , Escherichia coli 6346 G arma CTX-M ,0.25, arma CTX-M ,0.25, X1 arma CTX-M ,0.25, arma CTX-M ,0.25,0.25, X1 arma CTX-M ,0.25, NA NA NA NA NA NA NA NA NA 3552 arma CTX-M ,0.25, arma CTX-M ,0.25, arma CTX-M ,0.25, NA NA NA NA NA NA NA NA NA 3538 arma CTX-M ,0.25, NA NA NA NA NA NA NA NA NA 3558 arma CTX-M ,0.25, NA NA NA NA NA NA NA NA NA 3604 arma CTX-M ,0.25, NA NA NA NA NA NA NA NA NA 4356 I arma CTX-M ,0.25, arma CTX-M ,0.25, J arma CTX-M , arma CTX-M , Z arma CTX-M ,0.25, arma CTX-M-3 512,1.64,1,0.25, Bogaerts et al. NA, not analysed. Isolates were typed by PGE and one strain of each type was further analysed by conjugation experiment. MICs of amikacin (AMK), ceftazidime (CAZ), cefotaxime (CTX), cefepime (EP), meropenem (MER), ciprofloxacin (CIP) and sulfamethoxazole/trimethoprim (SXT) were determined. a MIC values for E. coli #4470: gentamicin, 512 mg/l; kanamycin and netilmicin, 256 mg/l; tobramycin, 128 mg/l. MIC values for all other isolates and transconjugants of gentamicin, kanamycin, netilmicin and tobramycin were.512 mg/l. Downloaded from at Pennsylvania State University on ebruary 27, 2014

5 Aminoglycoside 16S rrna methylases in Belgium were consistent with the PCR-sequencing results (not shown). All strains were found to co-produce CTX-M-3, CTX-M-14 or CTX-M-15, and 6 of 10 K. strains also harboured a TEM-3 ESBL, whereas a CMY-2 AmpC plasmid-borne b-lactamase was detected in one K.. Other b-lactamases also present in these isolates were TEM-1 in all 19 isolates and SHV-like in all K. isolates. PGE typing In order to delineate the clonality of the different isolates, all strains belonging to the same species were subjected to molecular typing (see igure 1). PGE analysis revealed two clusters of clonally related arma-positive K. (Table 2). The first cluster comprised five strains ( type) and the second comprised two strains (X1 type), suggesting that two small clonal outbreaks had occurred at one of the hospitals. On the contrary, all four arma-positive E. coli had distinct PGE patterns, suggesting horizontal spread of the resistance determinant. Conjugation experiments arma was transferable by conjugation with frequencies between 10 4 and 10 7 and was borne on the same plasmid as bla CTX-M and bla TEM genes (Table 2). Neither bla SHV from K. isolates nor bla CMY from K. #4356 were cotransferred suggesting that arma, bla SHV and bla CMY were probably carried by distinct genetic support. Resistance profiles and PCR analysis of the transconjugants from the two neomycinresistant E. coli (#3562 and #2312) selected on kanamycin confirmed that the strains harboured another plasmid with an aph(3 0 )-Ia gene (data not shown). (a) In contrast to the rmtb plasmid isolated by Doi et al., 9 the plasmid bearing rmtb was also self-transferable, as observed by Yan et al. 8 but bla CTX-M-14 was not co-transferred. Typing of plasmid incompatibility groups was performed on transconjugants and showed that, in the vast majority of the cases (11/13), arma was borne by IncL/M plasmids. In a previous work Galimand et al. showed that all arma-carrying human isolates analysed in European countries were indeed located on IncL/M plasmids. 13 We were not able to identify the plasmid incompatibility groups in two transconjugants originating from E. cloacae #3544 and E. coli #4461. The rmtb gene was associated with an IncI plasmid with the same inc/rep PCR profile as plasmid R These data further underscore the wide dissemination of 16S rrna methylases genes in several Enterobacteriaceae species of human origin as well as their location on various types of plasmids. To the best of our knowledge, this report constitutes the first detection of arma in Belgium and of an rmtb 16S rrna methylase gene in aminoglycoside-resistant Enterobacteriaceae in Europe. This retrospective study showed that the first occurrence of such resistant strains dated back to October 2000 for the arma-containing strains and October 2004 for that having rmtb. The importance of inter-country transfer of patients should also be highlighted since, in both hospitals, the index patients originated from foreign countries. Even though the occurrence of plasmid 16S rrna methylases in the two Belgian hospitals seems to be very low, this report further highlights the wide dissemination of this resistance mechanism among Enterobacteriaceae in a growing number of countries. The apparently higher prevalence of arma compared with that of rmtb could be due to its association on the same conjugative plasmid with the gene for CTX-M-3 and its location on functional transposon Tn The rapid spread of multidrug-resistant isolates Strain PGE type Z J X1 X1 I (b) Strain PGE type 4470 H 1540 C 4461 E 2312 D 6346 G igure 1. Dendrograms indicating the similarity (%) of PGE profiles of (a) K. and (b) E. coli clinical strains. 463

6 Bogaerts et al. producing both ESBLs and 16S rrna methylases raises clinical concern and may become a major therapeutic threat in the future. Other studies are required to determine the prevalence in other Belgian hospitals, to control further spread of the resistance. Acknowledgements We thank Professor Patrice Courvalin for his support and expert review of this manuscript. We are very grateful to Dr Alessandra Carattoli for providing us the controls for incompatibility group of plasmids. Preliminary results of this manuscript were presented at the orty-sixth Interscience Conference on Antimicrobial Agents and Chemotherapy, San rancisco, CA, 2006 (Abstract C2-64, p. 93). This work was funded in part by a grant from the Belgian Antibiotic Policy Coordination Committee (BAPCOC) of the Belgian Ministry for Public Health. Transparency declarations None to declare. References 1. Gilbert DN. Aminoglycosides. In: Mandell GL, ed. Principles and Practice of Infectious Diseases. 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