Departments of 1 Clinical Pathology and 2 Internal Medicine, College of Medicine, The Catholic University of Korea, Kangnam St Mary's Hospital, 505 Banpo-dong, Seocho-ku, Seoul, 137-701, Korea
Received 27 April 2004; returned 1 July 2004; revised 11 August 2004; accepted 29 September 2004
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Abstract |
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Methods: Over an 11 month period, a total of 12 P. mirabilis isolates showing resistance to ampicillin, gentamicin, ceftazidime, cefotaxime, cefuroxime, cefalothin, cefepime, piperacillin, trimethoprim/sulfamethoxazole and ciprofloxacin, were recovered from the sputum and urine specimens of nine patients who were hospitalized in the neurosurgery ward. The extended-spectrum ß-lactamases were screened with a double disc synergy test using ceftazidime, cefotaxime, aztreonam, cefepime and clavulanate. The ESBL types were determined by PCR using specific primers for blaTEM-1, blaSHV-1, blaCTX-M-1, blaCTX-M-2, blaCTX-M-8, blaCTX-M-9, blaPER-1, blaGES-1, blaVEB-1, blaOXA-10 and blaOXA-13 followed by sequencing. All the isolates underwent molecular typing by PFGE. The transferability was examined by conjugation.
Results and conclusions: All the isolates showed a marked synergy between the extended-spectrum cephalosporins and clavulanate together with an unusual synergy between cefoxitin and the cephalosporins (cefalothin, cefuroxime, ceftazidime, cefotaxime) and between imipenem, and ceftazidime and cefotaxime. Isoelectric focusing of the crude bacterial extracts showed a ß-lactamase band with a pI value of 5.4, which was inhibited by clavulanate. PCR and sequencing identified the gene to be blaVEB-1. In addition, the aadB gene was detected, conferring aminoglycoside resistance. The resistance was not transferred by conjugation. The outbreak was of a clonal origin as shown by PFGE demonstrating an identical banding pattern. This is the first report of VEB-1-producing Enterobacteriaceae in Korea.
Keywords: P. mirabilis , ESBLs , ß-lactam resistance , multidrug-resistant
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Introduction |
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Another non-TEM, non-SHV ESBL, VEB-1, has been described in Enterobacteriaceae3,4 and Pseudomonas aeruginosa from Southeast Asia,5 and Acinetobacter baumannii from France.6 However, to the best of our knowledge, the emergence of VEB-1 ESBL has not been reported in the Far East. This paper reports a nosocomial outbreak caused by VEB-1-producing P. mirabilis in a neurosurgery (NS) ward in Korea.
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Materials and methods |
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From October 2002 to August 2003, 12 non-duplicated (excluding the isolates from the same specimen from the same patients), multi-resistant P. mirabilis strains were recovered from nine patients who were hospitalized in the NS ward of the KangNam St Mary's Hospital in Seoul, Korea. These isolates were most frequently recovered from the respiratory tract (seven isolates) and urine (three isolates), but were also recovered from an open wound (one isolate) and a closed wound (one isolate). Bacterial identification was based on the results of the Vitek GNI card (bioMérieux Vitek Inc., Hazelwood, MO, USA) as well as conventional biochemical tests.
Antimicrobial susceptibility testing and screening for ESBL
The antibiotic susceptibility test was carried out using a disc diffusion test and the phenotypic confirmation test for ESBLs was carried out using a double disc synergy test according to the NCCLS guidelines.7 MICs of gentamicin, ciprofloxacin, cefuroxime, cefotaxime, ceftazidime, cefepime, ampicillin, cefoxitin, imipenem and meropenem were determined by an agar dilution method according to NCCLS guidelines.7
Isoelectric focusing
Crude ß-lactamase preparations, derived from the sonicated bacterial cultures of the P. mirabilis isolates, were assessed for the ß-lactamase pIs and the general inhibitor profile by isoelectric focusing (IEF). IEF was carried out at room temperature on a Bio-Rad mini isoelectric focusing III (Bio-Rad, Richmond, CA, USA). The enzymes were visualized by staining the gel with a 0.5 mM nitrocefin solution (BBL, Cockeysville, MD, USA). The isoelectric points of the enzymes from the P. mirabilis isolates were estimated by comparison with TEM-1, TEM-10, SHV-1, SHV-5 and CMY-1.
PCR amplification of ß-lactamase genes and sequencing
A search for the blaTEM-1, blaSHV-1, blaCTX-M-1, blaCTX-M-2, blaCTX-M-8, blaCTX-M-9, blaPER-1, blaGES-1, blaVEB-1, blaOXA-10 and blaOXA-13 genes in the clinical isolates was carried out by PCR amplification with the following sets of primers: TEM-1F, 5'-AAGCCATACCAAACGACGAG-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'-GGYYAAAAAATCACTGCGTC-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-8F, 5'-AGCAAAGTGAAACGCAAAAG-3' and CTX-8B, 5'-TCATTCGTCGTACCATAATC-3' for blaCTX-M-8; 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; VEB-1F, 5'-CGACTTCCATTTCCCGATGC-3' and VEB-1B, 5'-GGACTCTGCAACAAATACGC-3' for blaVEB-1; OXA-10F, 5'-TCTTTCGAGTACGGCATTAGC-3' and OXA-10B, 5'-CCAATGATGCCCTCACTTTCC-3' for blaOXA-10; and OXA-13F, 5'-ATTACTGCGTGTCTTTCA-3' and OXA-13B, 5'-CTCTTTCCCATTGTTTCA-3' for blaOXA-13.
In addition, the PCR amplifications were carried out with the arr-2 and aadB genes (conferring resistance to rifampicin and aminoglycosides, respectively) using the following sets of primers: 5'-ATATGCGGCCTAACAATTCG-3' and 5'-TCAAGCAACTCTGCGAGGA-3' for arr-2; and 5'-GACACAACGCAGGTCACATT-3' and 5'-CGCATATCGCGACCTGAAAGC-3' for aadB.
VEBcas-F (5'-GTTAGCGGTAATTTAACCAGATAG-3') and VEBcas-B (5'-CGGTTTGGGCTATGGGCAG-3'), located at each end of the blaVEB-1 cassette, were used to amplify the entire blaVEB-1 gene. A combination of 5'-CS or 3'-CS primers and VEBINV1 (5'-CAGTTTGAGCATTTGAATACAC-3') or VEBINV2 (5'-AGCGTATTTGTTGCAGAGTCC-3'), respectively, both primers reading outwards from blaVEB-1, was also used for the determination of the genetic content of class 1 integron.
The freshly isolated colonies were suspended in distilled water and adjusted to a turbidity equivalent to that of a 0.5 McFarland standard, and then boiled for 10 min. The supernatant, which was obtained after centrifugation at 12 000 rpm for 10 min, was used as template DNA. PCRs were carried out in 50 µL volumes containing 50 ng of DNA, 25 pM of each primer, 100 µM dNTPs, 2.5 U of Takara Ex Taq (Takara, Shiga, Japan) and the PCR buffer, with the following parameters: 94°C for 10 min; 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 3 min; and a final extension at 72°C for 10 min. For direct DNA sequencing, PCR products of the blaVEB-1-like gene, the blaTEM-1-like gene and the integron content (primers 5'-CS or 3'-CS and VEBINV1 or VEBINV2) were purified with a Qiaquick PCR purification kit (Qiagen, Hilden, Germany) and sequencing reactions were carried out with an automated sequencer (AI 377; Applied Biosystems, Foster City, CA, USA). Nucleotide sequence analysis and alignment methods were obtained from the National Center of Biotechnology Information Website (http://www.ncbi.nlm.nih.gov).
Conjugal transfer
Conjugal experiments were carried out several times between the clinical isolates and three recipients (sodium azide-resistant Escherichia coli J53, nalidixic acid-resistant E. coli RG 176, and rifampicin-resistant E. coli RG 488). The transconjugants were selected on a nutrient agar containing sodium azide (150 mg/L) or nalidixic acid (200 mg/L) or rifampicin (150 mg/L) plus either ceftazidime (2 mg/L) or cefotaxime (2 mg/L).
PFGE analysis
PFGE analysis was carried out according to the manufacturer's protocol (Bio-Rad). Briefly, the whole-cell DNA of the P. mirabilis isolate was digested with the SfiI restriction enzyme for 4 h at 50°C. Electrophoresis was carried out with a CHEF DRII (Bio-Rad) through a 1% agarose gel in 0.5x Tris/borate/EDTA buffer at 14°C, a voltage of 6 V/cm and a switch angle of 60°, using pulse times ranging from 5 to 50 s for 24 h. A bacteriophage -DNA ladder (Bio-Rad) was used as a DNA molecular weight marker.
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Results and discussion |
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In order to prevent the spread of this resistance gene, the characteristics of VEB-1 showing synergy between imipenem and the extended-spectrum cephalosporins, should aid in the detection and differentiation of VEB-1 from other class A ß-lactamases.
To the best of our knowledge, this is the first report of P. mirabilis producing VEB-1 ESBL in Far East Asia. The early recognition and rapid identification of the colonizing antimicrobial-resistant bacteria, including VEB-1-producing P. mirabilis, would be the most effective measures for coping with the further spread of this hazardous microorganism in clinical environments.
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Acknowledgements |
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Footnotes |
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References |
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