JAC Advance Access originally published online on November 10, 2005
Journal of Antimicrobial Chemotherapy 2006 57(1):156-158; doi:10.1093/jac/dki408
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Correspondence |
Occurrence of extended-spectrum ß-lactamases and plasmid-mediated AmpC ß-lactamases among Korean isolates of Proteus mirabilis
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 Seoul Medical Science Institute, Seoul Clinical Laboratories, Seoul, Korea; 3 Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea; 4 Korea Food and Drug Administration, Seoul, Korea; 5 Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Korea
* Corresponding author. Tel: +82-2-590-1604; Fax: +82-2-592-4190; E-mail: yjpk{at}catholic.ac.kr
Keywords: ESBLs , plasmids , P. mirabilis
Sir,
Proteus mirabilis is considered one of the most ß-lactam-susceptible members of the Enterobacteriaceae because it does not express a chromosomally encoded AmpC cephalosporinase. Although extended-spectrum ß-lactamases (ESBLs) have recently been identified in P. mirabilis isolates,1 there are few reports of plasmid-mediated AmpC ß-lactamase (PABL) in P. mirabilis, and the incidence was reported to be very low (0.4%) in a study carried out in the United States in the year 2000.2 Here we report a survey on ESBL and PABL production among clinical isolates of P. mirabilis from 12 nationwide clinical laboratories in Korea.
In a nationwide survey conducted between April and June 2004, a total of 134 consecutive P. mirabilis isolates were collected from 12 clinical laboratories. They were identified using either a Vitek GNI card (bioMérieux, Marcy-l'Etoile, France) or a Microscan GN combo card (Dade Behring, West Sacramento, CA, USA.). The MICs of cefalotin, cefuroxime, ceftazidime, cefotaxime, aztreonam, cefepime, cefoxitin, amikacin, gentamicin, piperacillin, ciprofloxacin, meropenem and imipenem were determined using the agar dilution method.3 ESBL detection was based on a double-disc-synergy test.4 The modified Hodge test5 was used to screen PABL.
For the ESBL screen-positive isolates, a search for the blaTEM-1, blaSHV-1, blaCTX-M-1, blaCTX-M-2, blaCTX-M-9, blaPER-1, blaGES-1 and blaVEB-1 genes was performed by PCR amplification with the following sets of primers: TEM-1F, 5'-AGCCATACCAAACGACGAG-3' and TEM-1B, 5'-ATTGTTGCCGGGAAGCTAGA-3' for blaTEM-1; SHV-1F, 5'-TATCCCTGTTAGCCACCCTG-3' and SHV-1B, 5'-CACTGCAGCAGCTGC(A/C)TT-3' for blaSHV-1; CTX-1F, 5'-GGTTTAAAAAATCACTGCGTC-3' and CTX-1B, 5'-TTGGTGACGATTTTAGCCGC-3' for blaCTX-M-1; CTX-2F, 5'-ATGATGACTCAGAGCATTCG-3' and CTX-2B, 5'-TGGGTTACGATTTTCGCCGC-3' for blaCTX-M-2; CTX-9F, 5'-CGCTTTATGCGCAGACGA-3' and CTX-9B, 5'-GATTCTCGCCGCTGAAGC3' for blaCTX-M-9; PER-1F, 5'-ATGAATGTCATTATAAAAGC-3' and PER-1B, 5'-AATTTGGGCTTAGGGCAGAG-3' for blaPER-1; GES-1F, 5'-ATGCGCTTCATTCACGCAC-3' and GES-1B, 5'-CTATTTGTCCGTGCTCAGG-3' for blaGES-1; and VEB-1F, 5'-CGACTTCCATTTCCCGATGC-3' and VEB-1B, 5'-GGACTCTGCAACAAATACGC-3' for blaVEB-1.
For the isolates with cefoxitin MICs of 
16 mg/L, plasmids were isolated using a Wizard Plus SV Miniprep DNA purification system (Promega, MD, USA) and PABL amplification was carried out using multiplex PCR which can detect various types (MOX, CMY, LAT, DHA, ACC, MIR-1, ACT-1 and FOX) of PABLs.6 All the sequencing reactions were performed using an automated sequencer (AI 377; Applied Biosystems, CA, USA). The genomic DNA for PFGE analysis was digested with SfiI (New England Biolabs, Beverly, MA, USA).
Of the 134 P. mirabilis isolates, 39 (29.1%) were cefalotin resistant (MICs 32 mg/L) and 24 (17.9%) were found to be cefuroxime resistant (MICs 32 mg/L) according to the NCCLS guideline (M100-S15). Thirteen (9.7%) isolates showed some potentiation of the inhibitory zones of the ß-lactams by clavulanic acid, suggesting the presence of ESBL activity. From these 13 putative ESBL-positive isolates, the structural genes for blaCTX-M-9-like, blaTEM, blaSHV and blaPER-1 were found either alone or in combination (Table 1). Sequencing analysis identified the ESBL genes blaCTX-M-14, blaTEM-52, blaSHV-12 and blaPER-1. blaCTX-M-14 type, which is one of the CTX-M-9 group enzymes and has one amino acid change at position 231 from blaCTX-M-9, was the most frequently encountered ESBL. Most of the ESBL producers also harboured blaTEM-1. All but one of the ESBL-producing strains was resistant to gentamicin, and 8 (61.5%) of the 13 ESBL producers were resistant to amikacin. Of the ESBL-negative isolates, gentamicin resistance was observed in 26/121 (21.5%) and amikacin resistance was observed in only 2/121 (1.7%). The rate of ciprofloxacin resistance was also higher in the ESBL producers (8/13, 61.5%) than in the ESBL non-producers (21/121, 17.4%). However, all the ESBL producers were susceptible to carbapenems. The eight blaCTX-M-14-positive isolates showed seven different PFGE patterns, indicating the presence of many clones. Moreover, the two blaPER-1 producers also showed different PFGE patterns (data not shown).
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Of the 134 P. mirabilis isolates, 15 isolates were not susceptible to cefoxitin and 11 of them were not susceptible to cefuroxime. Five isolates had a positive reaction according to the modified Hodge test; all were not susceptible to cefoxitin and cefuroxime. PCR and sequencing analysis revealed that four of them harboured PABL genes (two contained CMY-10, one CMY-2 and one DHA-1), but in one isolate no PABL gene was detected (Table 1). Of them, one of the CMY-10 harbouring isolates also harboured blaPER-1 and blaTEM-52. In contrast to general knowledge regarding the susceptibility pattern of the PABL producers, which are known to be resistant to cephalosporins in the oxyimino and 7-
-methoxy groups, this study found the DHA-1 producer to be susceptible to ceftazidime and cefotaxime (both MICs 
1 mg/L), and the two isolates that produced only CMY-2 or CMY-10 to be susceptible to ceftazidime (MICs of 4 and 1 mg/L, respectively). This is in line with the finding that several AmpC-producing Escherichia coli showed variable MICs of ceftazidime and cefotaxime (0.5–128 mg/L and 0.5–16 mg/L, respectively).2 Therefore, the potential exists for PABL production to be missed in screens of P. mirabilis or E. coli using a criterion of MIC 2 mg/L for the oxyimino-group of cephalosporins. In this study, although the number of PABL producers was very small, of the 11 isolates that were not susceptible to both cefuroxime and cefoxitin, 4 isolates were PABL producers. In conclusion, this study found that the prevalence of ESBLs and PABLs in P. mirabilis was 9.7 and 3.0%, respectively. Further study is needed to investigate the screening criteria of PABL production in P. mirabilis.
Transparency declarations
None to declare.
Acknowledgements
We thank all the contributing laboratories that provided isolates for this study. We also thank Jin Kyung Yu for his excellent technical assistance and Hyun Jeong for the testing of isolates by PFGE. This work was supported by a grant from the Korea Food and Drug Administration in 2004 (FD100-04062).
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