JAC Advance Access originally published online on August 4, 2006
Journal of Antimicrobial Chemotherapy 2006 58(4):907-908; doi:10.1093/jac/dkl317
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Correspondence |
Co-production of 16S rRNA methylases and extended-spectrum ß-lactamases in AmpC-producing Enterobacter cloacae, Citrobacter freundii and Serratia marcescens in Korea
1 Department of Clinical Pathology, College of Medicine, The Catholic University of Korea, Kangnam St Mary's Hospital 505 Banpo-dong, Seocho-ku, Seoul, 137-701, Korea 2 Department of Clinical Pathology, College of Medicine, The Catholic University of Korea, Holy Family Hospital 2 Sosa-dong, Wonmi-gu, Pucheon, Kyunggi-do, 420-717, Korea 3 Korea Food and Drug Administration, 231 Jinheungno, Eunpyeong-gu, Seoul 122-704, Korea 4 Department of Laboratory Medicine, Yonsei University College of Medicine 134 Sinchon-dong, Seodaemun-ku, Seoul, 120-752, Korea 5 Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases, 4-7-1 Gakuen Musashi-Murayama Tokyo 208-0011, Japan
*Corresponding author. Tel: +82-2-590-1604; Fax: +82-2-592-4190; E-mail: yjpk{at}catholic.ac.kr
Keywords: ESBLs , E. cloacae , C. freundii , S. marcescens
Sir,
Enterobacter cloacae, Citrobacter freundii and Serratia marcescens are important nosocomial pathogens. In Korea, extended-spectrum ß-lactamase (ESBL) prevalence in AmpC-producing E. cloacae, C. freundii and S. marcescens is quite high (10.9–23.6%), and these ESBL-producers show higher resistance rates to aminoglycosides than do the ESBL-non-producers.1 While ribosomal protection mediated by methylation of 16S rRNA has been known as a self-defense mechanism for aminoglycoside-producing actinomycetes, it was not reported in other species until 2003. However, a series of methylases have been identified in several nosocomial pathogens, including Pseudomonas aeruginosa,2 S. marcescens,3 Proteus mirabilis4 and Klebsiella pneumoniae.5 The existence of these enzymes (RmtA, RmtB, RmtC and ArmA) is of great concern because they are capable of conferring an extraordinary high level of resistance (MIC > 512 mg/L) against most clinically important aminoglycosides, and they were often associated with ESBLs.6
In the present study, a total of 413 consecutive, non-duplicate isolates, including E. cloacae (158), C. freundii (126) and S. marcescens (129), were collected during March–July 2003 at 11 university hospitals in Korea. The isolates were from wound (37%), urine (35%), respiratory specimen (20%), blood (4%) and body fluid (4%). The MICs of amikacin (8–512 mg/L) and arbekacin (8–512 mg/L) were determined by an agar dilution method in accordance with the CLSI guideline. The detection of ESBL was based on the double disc synergy test (DDST) using discs containing 30 µg of ceftazidime, cefotaxime, aztreonam and cefepime. They were placed 2 cm from a disc containing amoxicillin/clavulanic acid (20/10 µg) (BBL, Cockeysville, MD, USA).
For the isolates that showed high-level resistance (MICs of >512 mg/L) to amikacin or arbekacin, a search for the 16S rRNA methylase genes (rmtA, rmtB, rmtC and armA) was performed by PCR. The total DNAs were extracted from isolates by boiling and the PCR was carried out with the Taq DNA polymerase (Takara Shuzo, Shiga, Japan) and the following sets of primers: rmtA-F, 5'-CTA GCG TCC ATC CTT TCC TC-3'; rmtA-R, 5'-TTT GCT TCC ATG CCC TTG CC-3'; rmtB-F, 5'-CCC AAA CAG ACC GTA GAG GC-3'; rmtB-R, 5'-CTC AAA CTC GGC GGG CAA GC-3'; rmtC-F, 5'-CGA AGA AGT AAC AGC CAA AG-3'; rmtC-R, 5'-ATC CCA ACA TCT CTC CCA CT-3'; armA-F, 5'-AGG TTG TTT CCA TTT CTG AG-3'; armA-R, 5'-TCT CTT CCA TTC CCT TCT CC-3'. Plasmids harbouring each gene were used as positive controls. For the detection of the ESBL genes, primers specific for blaTEM (TEM-F, 5'-ATA AAA TTC TTG AAG AAA-3'; TEM-R, 5'-GAC AGT TAC CAA TGC TTA ATC-3'), blaSHV (SHV-F, 5'-TGG TTA TGC GTT ATA TTC GCC-3'; SHV-R, 5'-GGT TAG CGT TGC CAG TGC T-3'), blaCTX-M (CTX-M-F, 5'-CGC TTT GCG ATG TGC AG-3'; CTX-M-R, 5'-ACC GCG ATA TCG TTG GT-3'), blaCTX-M-9 (CTX-M-9-F, 5'-CGC TTT ATG CGC AGA CGA-3'; CTX-M-9-R, 5'-GAT TCT CGC CGC TGA AGC-3') and blaPER-1 (PER-1-F, 5'-AAT TTG GGC TTA GGG CAG AA-3'; PER-1-R, 5'-ATG AAT GTC ATT ATA AAA GC-3') were used. PCR products were purified with a QIAquick PCR purification kit (QIAGEN, Hilden, Germany) and sequenced on a 3730 DNA analyser (Applied Biosystems, Foster City, CA, USA). The nucleotide and deduced protein sequences were analysed with software available from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov).
Of the total 413 isolates, 58 were resistant to amikacin, and the majority (49/58, 84.5%) of these 58 isolates showed high-level resistance (MIC > 512 mg/L) to both amikacin and arbekacin. One E. cloacae isolate was highly resistant to arbekacin (MIC > 512 mg/L), but it was susceptible to amikacin (16 mg/L). The frequency of the high-level resistance to amikacin or arbekacin was 9.5% (15/158), 10.3% (13/126) and 17.1% (22/129) for E. cloacae, C. freundii and S. marcescens isolates, respectively, and almost all of them (13 E. cloacae, 12 C. freundii and 21 S. marcescens isolates) harboured the armA gene. One C. freundii isolate harboured the rmtB gene. The 16S rRNA methylase-harbouring isolates were isolated from nine hospitals distributed nationwide. All of them were highly resistant to both arbekacin and amikacin. None harboured an rmtA or rmtC gene. The ESBL production rate was significantly higher in 16S rRNA methylase-producers (100%, 76.9% and 66.7% among E. cloacae, C. freundii and S. marcescens, respectively), compared with 16S rRNA methylase-non-producers (25.0%, 12.4% and 10.2%, respectively) (P = 0.002, P < 0.001 and P < 0.001, respectively). Most of the ArmA producers co-harboured various ESBLs (CTX-M-3, CTX-M-9, CTX-M-14, TEM-52 and SHV-12), among which CTX-M-3 was the most common (Table 1).
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This finding corroborates the previous reports that the armA was frequently associated with blaCTX-M and they were co-transferred by conjugation.6,7 Although only one isolate harboured the rmtB gene in the present study, it also harboured CTX-M-14; this coincides with the report by Yan et al.6 where six out of the seven rmtB-positive isolates harboured CTX-M-14. In Korean medical practice, arbekacin is only rarely used for the treatment of methicillin-resistant Staphylococcus aureus. Nevertheless, the prevalence of high-level resistance to amikacin and arbekacin was similarly high, suggesting that armA can confer resistance to arbekacin as in other 16S rRNA methylases.2,3
In conclusion, the armA gene is widespread in Korean isolates of E. cloacae, C. freundii and S. marcescens, and an rmtB producer was also found. Given the multiresistance in these isolates, prudent antibiotic use, accurate detection of this resistance and strict infection control are urgently needed to prevent the spread of these organisms.
Transparency declarations
None to declare.
Acknowledgements
We thank all the contributing laboratories that provided isolates for the study. We also thank Jung Jun Park and Kyongho Cha for excellent technical assistance. This work was supported by a grant from the Korea Food and Drug Administration in 2005 (FD100-05062).
References
1 Park YJ, Lee S, Yong D, et al. (2003) Antimicrobial susceptibility of inducible AmpC ß-lactamase-producing Enterobacter cloacae, Citrobacter freundii, and Serratia marcescens: a Korean survey. Kor J Lab Med 23:251–7.
2 Yokoyama K, Doi Y, Yamane K, et al. (2003) Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa. Lancet 362:1888–93.[CrossRef][Web of Science][Medline]
3
Doi Y, Yokoyama K, Yaname K, et al. (2004) Plasmid-mediated 16S rRNA methylase in Serratia marcescens conferring high-level resistance to aminoglycosides. Antimicrob Agents Chemother 48:491–6.
4
Wachino J, Yamane K, Shibayama K, et al. (2006) Novel plasmid-mediated 16S rRNA methylase, RmtC, found in a Proteus mirabilis isolate demonstrating extraordinary high-level resistance against various aminoglycosides. Antimicrob Agents Chemother 50:178–84.
5
Galimand M, Courvalin P, Lambert T. (2003) Plasmid-mediated high-level resistance to aminoglycosides in Enterobacteriaceae due to 16S rRNA methylation. Antimicrob Agents Chemother 47:2565–71.
6
Yan JJ, Wu JJ, Ko WC, et al. (2004) Plasmid-mediated 16S rRNA methylases conferring high-level aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae isolates from two Taiwanese hospitals. J Antimicrob Chemother 54:1007–12.
7
Galimand M, Sabtcheva S, Courvalin P, et al. (2005) Worldwide disseminated armA aminoglycoside resistance methylase gene is borne by composite transposon Tn1548. Antimicrob Agents Chemother 49:2949–53.
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