Nosocomial outbreak by Proteus mirabilis producing extended-spectrum ß-lactamase VEB-1 in a Korean university hospital

Ja-Young Kim1, Yeon-Joon Park1,*, Sang-Il Kim2, Moon Won Kang2, Seung-Ok Lee1 and Kyo-Young Lee1

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Objectives: To examine the molecular mechanisms involved in the ß-lactam resistance of multidrug-resistant Proteus mirabilis isolates that showed an unusual synergy between imipenem and ceftazidime in a Korean hospital.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
While most Proteus mirabilis isolates are susceptible to ß-lactams, some isolates produce extended-spectrum ß-lactamases (ESBLs).1 Clinical occurrences of ESBL-producing P. mirabilis strains have increased since ESBL production in P. mirabilis was first documented in 1987. In France, the prevalence of ESBL-positive isolates has increased from 0.8% of P. mirabilis isolates in 1991 to 6.9% in 1998. Surveillance studies conducted in the United States and Italy showed a 9.5% and 8.8% ESBL prevalence among P. mirabilis isolates.1 Although the most predominant plasmid-mediated ß-lactamases found in the clinical isolates of P. mirabilis are TEM-derived ESBLs, such as TEM-8, TEM-10, TEM-21, TEM-26 and TEM-66,1 non-TEM, non-SHV derivatives, such as CMY-3, CMY-4, CEP-1, CTX-M-2 and PER-2, have also been reported in specific geographical distributions.2

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Bacterial isolates

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 {lambda}-DNA ladder (Bio-Rad) was used as a DNA molecular weight marker.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
This work began with the observation that a P. mirabilis clinical isolate showed an unusual synergy between imipenem and cephalosporins (ceftazidime and cefotaxime), which is suggestive of an unusual ß-lactamase. During a particular period, a total of 12 isolates showing an identical antibiogram were isolated (Table 1). The double disc synergy test was positive showing a marked synergy between ceftazidime or cefotaxime and clavulanate together with an unusual synergy between cefoxitin and cephalosporins (cefalothin, cefuroxime, ceftazidime, cefotaxime) and between imipenem, and cefotaxime and ceftazidime. Isoelectric focusing showed a ß-lactamase band of pI 5.4, which was inhibited by clavulanate.1,3 The PCR search revealed that all the P. mirabilis isolates contained amplification products for the blaVEB-1-like gene and the blaTEM-1-like gene, whereas no amplicons were produced for the other primers. Sequence analysis of the whole ORF of blaVEB-1-like gene and blaTEM-1 PCR products revealed a 100% homology with that of blaVEB-1b (AF324834) and blaTEM-1,1,2 respectively. Using 5'-CS, 3'-CS and VEBINV 1/2 primers, PCR fragments were obtained, indicating that blaVEB-1 was part of a class 1 integron.


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Table 1. MICs of various antibiotics for P. mirabilis strain

 
VEB-1 was first reported in the clinical isolates of E. coli and Klebsiella pneumoniae from a Vietnamese patient in France in 1998.3 VEB-1 has a molecular mass of approximately 30 kDa and it belongs to the Ambler class A ß-lactamases and to the Bush group 2be.3 It shares the highest sequence identity (38%) with PER-1 and PER-2, which are mainly found in A. baumannii, and a significant sequence identity with CBLA and CEPA in Bacteroides spp.3,8 It confers high-level resistance to a broad spectrum of cephalosporins. However, this activity is inhibited not only by clavulanate, but also by cefoxitin and imipenem.1 A similar resistance and inhibitor profile were observed with the PER-1 and GES-1 positive strains,8,9 where the synergy pattern is more difficult to detect with the GES enzyme.9 Unlike most of the class A ESBL genes, blaVEB-1, together with blaGES-1, form part of an integron,3,9 and are associated with the oxa-10, arr-2, or aadB gene cassettes. In this study, the isolates harboured the aadB gene, but not the oxa-10 or arr-2 genes. Transfer by conjugation of either ceftazidime or cefotaxime resistance failed, which suggests that it is not located on a conjugative plasmid and it coincides with the report that blaVEB-1b was chromosomally located.10 The PFGE profiles of the SfiI-restricted DNA of all P. mirabilis isolates were identical, indicating the clonal spread of the strain within the hospital (Figure 1).



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Figure 1. PFGE banding patterns after SfiI digestion of the representative VEB-1-positive P. mirabilis isolates. Lane M, {lambda}-DNA ladder; lanes 1–10, clinical isolates of P. mirabilis.

 
The blaVEB-1 gene was found in four different bacterial species (E. coli, K. pneumoniae, P. aeruginosa, P. mirabilis) from patients in the same geographical location (Southeast Asia).35 This finding suggests that the blaVEB gene can spread among clinically relevant species,35 and there is considerable heterogeneity of the genetic environment in which the blaVEB alleles are found in different clinical isolates.

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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
We thank Professor Kyungwon Lee and Jong Hwa Yum for helpful discussions, and Jung Jun Park and Sun Young Park for technical assistance.


    Footnotes
 
* Corresponding author. Tel: +82-2-590-1604; Fax: +82-2-592-4190; Email: yjpk{at}catholic.ac.kr


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
1 . Luzzaro, F., Perilli, M., Amicosante, G. et al. (2001). Properties of multidrug-resistant, ESBL-producing Proteus mirabilis isolates and possible role of ß-lactam/ß-lactamase inhibitor combinations. International Journal of Antimicrobial Agents 17, 131–5.[CrossRef][ISI][Medline]

2 . Naas, T., Benaoudia, F., Massuard, S. et al. (2000). Integron-located VEB-1 extended-spectrum ß-lactamase gene in a Proteus mirabilis clinical isolate from Vietnam. Journal of Antimicrobial Chemotherapy 46, 703–11.[Abstract/Free Full Text]

3 . Poirel, L., Naas, T., Guibert, M. et al. (1999). Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum ß-lactamase encoded by an Escherichia coli integron gene. Antimicrobial Agents and Chemotherapy 43, 573–81.[Abstract/Free Full Text]

4 . Girlich, D., Poirel, L., Leelaporn, A. et al. (2001). Molecular epidemiology of the integron-located VEB-1 extended-spectrum ß-lactamase in nosocomial enterobacterial isolates in Bangkok, Thailand. Journal of Clinical Microbiology 39, 175–82.[Abstract/Free Full Text]

5 . Naas, T., Poirel, L., Karim, A. et al. (1999). Molecular characterization of In50, a class 1 integron encoding the gene for the extended-spectrum ß-lactamase VEB-1 in Pseudomonas aeruginosa. FEMS Microbiology Letters 176, 411–9.[CrossRef][ISI][Medline]

6 . Poirel, L., Menuteau, O., Agoli, N. et al. (2003). Outbreak of extended-spectrum ß-lactamase VEB-1-producing isolates of Acinetobacter baumannii in a French hospital. Journal of Clinical Microbiology 41, 3542–7.[Abstract/Free Full Text]

7 . National Committee for Clinical Laboratory Standards. (2002). Performance Standards for Antimicrobial Disc Susceptibility Tests—Eighth Edition: Approved Standard M2-A8. NCCLS, Villanova, PA, USA.

8 . Luzzaro, F., Mantengoli, E., Perilli, M. et al. (2002). Dynamics of a nosocomial outbreak of multidrug-resistant Pseudomonas aeruginosa producing the PER-1 extended-spectrum ß-lactamase. Journal of Clinical Microbiology 39, 1865–70.[CrossRef][ISI]

9 . Poirel, L., Le Thomas, I., Naas, T. et al. (2000). Biochemical sequence analyses of GES-1, a novel class A extended-spectrum ß-lactamase, and the class 1 integron In52 from Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 44, 622–32.[Abstract/Free Full Text]

10 . Poirel, L., Rotimi, V. O., Mokaddas, E. M. et al. (2001). VEB-1-like extended-spectrum ß-lactamases in Pseudomonas aeruginosa, Kuwait. Emerging Infectious Diseases 7, 468–70.[ISI][Medline]