AAC Accepts, published online ahead of print on 2 June 2008 Antimicrob. Agents Chemother. doi: /aac

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1 AAC Accepts, published online ahead of print on June 00 Antimicrob. Agents Chemother. doi:.11/aac Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. VIM-15 and VIM-1, two new VIM--like Metallo-β-lactamases in Pseudomonas aeruginosa from Bulgaria and Germany Ines Schneider 1, Emma Keuleyan, Rudolf Rasshofer 3, Rumyana Markovska, Anne Marie Queenan 5, Adolf Bauernfeind 1 1 MICOER-Institute, Munich, Germany; Medical Institute, Ministry of the Interior, Sofia, Bulgaria; 3 Medizinisches Versorgungszentrum Labor Dr. Tiller & Partner, Munich, Germany; Department of Microbiology, Medical University, Sofia, Bulgaria; 5 Johnson & Johnson Pharmaceutical Research and Development, Raritan Running title: NJ, USA VIM-15 and VIM-1, new Metallo-β-lactamases in P. aeruginosa Corresponding author: Adolf Bauernfeind MICOER-Institute Hesseloherstrasse, 00 Munich, Germany Phone + Fax: bauernfeind@aol.com 1

2 ABSTRACT Two Pseudomonas aeruginosa urine isolates from Bulgaria and Germany produced two new VIM- variants. VIM-15 had one amino acid substitution (Tyr1Phe), which caused a significant increase in hydrolytic efficiency. The substitution Ser5Leu, characterizing VIM-1, showed no influence on enzyme activity. Both genes were part of class I integrons located in the chromosome.

3 VIM-type β-lactamases are common acquired metallo-β-lactamases (MBLs) mostly found in Pseudomonas aeruginosa (). They contribute significantly to the resistance of non-fermenting gram-negative organisms to carbapenems. Furthermore, as all bla VIMs are part of integrons, their acquisition is often linked with resistance to other compounds, e.g. aminoglycosides. We analyzed the MBLs of two P. aeruginosa strains isolated in Bulgaria and Germany. (Part of this work was presented at the 17 th ECCMID, Munich, Germany, 007 [abstr. O95].) P. aeruginosa 1301 was isolated in April 005 at the Medizinisches Versorgungszentrum, Munich, Germany, from urine of a 7-year old, male patient. P. aeruginosa 9551 was recovered in March 00 at the Medical Institute, Ministry of the Interior, Sofia, Bulgaria, from urine of a 9-year old, male patient treated at the nephrology ambulatory. MICs, determined by the agar dilution technique following Clinical and Laboratory Standards Institute guidelines (), are shown in Table 1. Both P. aeruginosa strains were resistant to carbapenems suggesting the presence of carbapenemases. A Hodge test and a double-disk-synergy-test with EDTA, carried out as described (5), demonstrated the production of carbapenem-hydrolyzing enzymes susceptible to EDTA inhibition, thereby confirming the presence of MBLs. A PCR with bla VIM -specific oligonucleotides (VIM-F, 5 - TTGGTCGCATATCGCAAC-3 ; VIM-R, 5 -CGCAGCACCRGGATAGAA-3 ) was positive. We used combinations of VIM-F and VIM-R with oligonucleotides binding to conserved regions of class I integrons (qace 1, 5 -GCCAACTATTGCGATAAC- 3 ; IntIa-attI, 5 -TCTATGCCTCGGGCATCC-3 ) to sequence the whole gene and its environment. The sequences revealed close homology to bla VIM-, although one 3

4 nucleotide substitution causing an amino acid substitution in comparison to VIM was found for both strains. The nucleotide substitution A5T leading to the tyrosine 1 phenylalanine substitution characterized the VIM-enzyme of P. aeruginosa 9551 (MBL-numbering according to Garau et al.)(). At that position, all VIM-type MBLs described so far have a tyrosine, except VIM-7, which also shows a phenylalanine but shares only 7% amino acid identity with VIM-. The bla VIM of P. aeruginosa 1301 had one nucleotide substitution (C1T), causing the serine 5 leucine substitution. At that position, all other VIM-variants carry a serine. These new VIM- type MBLs were named VIM-15 (P. aeruginosa 9551) and VIM-1 (P. aeruginosa 1301). To explore whether the amino acid substitutions influence hydrolytic activity, we cloned the bla VIM genes of P. aeruginosa 9551, 1301 and of the VIM- producing reference strain P. aeruginosa 9//U1315 in an isogenic background. Cloning was performed as described using the vector pbc and Escherichia coli DH5α (). Primers VIM--EcoRI-V (5 -AGGAATTCCTAGTGCCGCACTCACC-3 ) and VIM-- BamHI-R (5 -CAGGATCCTTCATGTTATGCCG-3 ) amplifying the gene, including 7 bp of the upstream and 1 bp of the downstream region, were used. The expression of the bla VIM -genes in the transformants led to a significant increase in MICs for amoxicillin ( times) and cefotaxime (3-1 times), and to a moderate increase in MICs for piperacillin/tazobactam and ceftazidime (- times) (Table 1). The MICs of cefepime ( times), meropenem ( times), imipenem (1- times), and aztreonam were not or only slightly affected. The lower MICs for ceftazidime in comparison to values found in the literature (7) were caused by the absence of the original promoter, which was removed during cloning. Interestingly, the production of VIM-15 caused slightly higher MICs for cefotaxime and imipenem than VIM- or VIM-1. Increased activity

5 of VIM-15 in comparison to VIM- and VIM-1 against cefotaxime, meropenem and imipenem could be seen by a Hodge test using crude strain homogenates (data not shown). Purification of β-lactamases from transformants producing VIM-15 and VIM-1 and determination of kinetic parameters were carried out as described (9). Overnight cultures were centrifuged, washed with phosphate buffer and subjected to five freeze- thaw cycles. Following centrifugation supernatants were filtered and passed through a Superdex 0 gel filtration column. Active fractions were further purified using HiTrap SP cation and Q anion-exchange columns. The data for VIM-, previously obtained using the same procedure, were taken from Queenan et al. (9). Both VIM-15 and VIM-1 showed a higher turnover rate ( ) for cefotaxime than for ceftazidime and cefepime, resulting in higher hydrolytic efficiencies ( /K m ) for cefotaxime (Table ). All three enzymes hydrolyzed imipenem faster than meropenem and, typical for MBLs, aztreonam was hydrolyzed only very slowly. VIM-1 presented and K m values very similar to those for VIM-, resulting in similar hydrolytic efficiencies. The amino acid substitution from serine to leucine at position 5 is located on the second β-strand of the enzyme (3). Although this is a non-conservative change, because of its remote position in relation to the active site no influence on the hydrolytic activity is expected. VIM-15 showed hydrolytic efficiencies about one order of magnitude higher than those of VIM- and VIM-1 for all substrates tested. This is caused by lower K m values for cephaloridine, cefotaxime, imipenem, and meropenem, indicating a stronger affinity of VIM-15 for those substrates. In contrast, the increased activity for cefepime was caused by an elevated turnover rate. The higher efficiencies for ceftazidime and benzylpenicillin were a result of both increased turnover rate and 5

6 decreased K m values. The substitution from tyrosine to phenylalanine at position is located on β-strand 11 (3) and has a conservative character. However, this substitution is near the cysteine zinc binding site (Cys1) and apparently affected the binding properties or catalytic activity of VIM-15. Both, bla VIM-15 and bla VIM-1, were part of class I integrons. The integron of P. aeruginosa 9551 harbored the bla VIM-15 cassette only. This structure is identical to that of the VIM- producing P. aeruginosa COL-1 isolated in 199 in France (7). The bla VIM-1 cassette of P. aeruginosa 1301 was flanked by two aac( )-Ib cassettes coding for an aminoglycoside acetyltransferase. This structure is identical to that of the VIM- producing P. aeruginosa B330 isolated in 003 in Germany except that the integron of P. aeruginosa B330 harbored additionally cmla and ant(3 )-Ib cassettes (). For P. aeruginosa 9551, the absence of cassettes coding for aminoglycoside modifying enzymes is in accordance with the susceptibility to aminoglycosides while P. aeruginosa 1301, harboring two genes of an aminoglycoside acetyltransferase, was highly resistant to aminoglycosides (Table 1). To test whether the bla VIM containing integrons reside on plasmids, we tried their transfer by conjugation to E. coli C00 R - (1) and electroporation to E. coli DH5α. Both attempts failed. The bla VIM localization was further analyzed by a Southern blot of plasmid preparations (Qiagen Plasmid Midi Kit, Qiagen, Hilden, Germany) followed by hybridization with probes amplified from bla VIM (VIM-F; VIM-- BamHI-R) and 1S rrna (1V, 5 -AGAGTTTGATCMKGGCTCAG-3 ; R, 5 - CAGGATCCTTCATGTTATGCCG-3 ) using the Gene Images AlkPhos Direct Labeling and Detection System (GE Healthcare, Little Chalfont, UK). In addition, we treated plasmid DNA preparations with Plasmid-Safe (Epicentre Biotechnologies, Madison, USA), a DNase which selectively digests linear DNA but not circular DNA.

7 For both P. aeruginosa strains, a DNA band which was recognized by the VIM probe 1 1 as well as by the 1S rrna probe and which was affected by treatment with Plasmid- Safe was found. Assuming those bands as chromosomal DNA, the bla VIM genes appear to be chromosomally located. In conclusion, the isolation of two new VIM-type MBLs in Bulgaria and Germany highlights the ongoing spread and evolution of this group of β-lactamases. VIM-type MBLs have already been described in Germany (,11), although no report on VIM- MBLs in Bulgaria was found. While the Ser5Leu substitution has no influence on hydrolytic activity, the amino acid substitution from tyrosine to phenylalanine at position 1 enhances enzymatic activity. The nucleotide sequences of bla VIM-15 and bla VIM-1 will appear in the GenBank database under accession numbers EU1975 and EU197. We thank Wenchi Shang, Johnson & Johnson Pharmaceutical Research and 1 Development, Raritan NJ, USA, for purification of the VIM β-lactamases and Yunsop Chong and Kyungwon Lee, Yonsei University College of Medicine, Seoul, Korea, for providing us with the VIM- reference strain P. aeruginosa 9//U

8 REFERENCES Bauernfeind, A., S. Wagner, R. Jungwirth, I. Schneider, and D. Meyer A novel class C β-lactamase (FOX-) in Escherichia coli conferring resistance to cephamycins. Antimicrob. Agents Chemother. 1: Garau, G., I. García-Sáez, C. Bebrone, C. Anne, P. Mercuri, M. Galleni, J. M. Frère, and O. Dideberg. 00. Update of the standard numbering scheme for class B β-lactamases. Antimicrob. Agents Chemother. : Garcia-Saez, I., J. D. Docquier, G. M. Rossolini, and O. Dideberg. 00. The three-dimensional structure of VIM-, a Zn-β-lactamase from Pseudomonas aeruginosa in its reduced and oxidised form. J. Mol. Biol. 375: Henrichfreise, B., I. Wiegand, K. J. Sherwood, and B. Wiedemann Detection of VIM- metallo-β-lactamase in Pseudomonas aeruginosa from Germany. Antimicrob. Agents Chemother. 9: Lee, K., Y. Chong, H. B. Shin, Y. A. Kim, D. Yong, and J. H. Yum Modified Hodge and EDTA-disk synergy tests to screen metallo-β-lactamaseproducing strains of Pseudomonas and Acinetobacter species. Clin. Microbiol. Infect. 7:-91.. National Center for Clinical and Laboratory Standards. 00. Performance standards for antimicrobial susceptibility testing, 1 th informational supplement. M0-S1. National Center for Clinical and Laboratory Standards, Wayne, Pa. 7. Poirel, L., T. Naas, D. Nicolas, L. Collet, S. Bellais, J. D. Cavallo, and P. Nordmann Characterization of VIM-, a carbapenem-hydrolyzing metallo-β-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Antimicrob. Agents Chemother. :91-97.

9 . Schneider, I., R. Markovska, E. Keuleyan, M. Sredkova, K. Rachkova, I. 1 Mitov, and A. Bauernfeind Dissemination and persistence of a plasmidmediated TEM-3-like β-lactamase, TEM-139, among Enterobacteriaceae in Bulgaria. Int. J. Antimicrob. Agents. 9: Queenan, A. M., W. Shang, M. Kania, M. G. P. Page, and K. Bush Interactions of ceftobiprole with β-lactamases from molecular classes A to D. Antimicrob. Agents Chemother. 51: Walsh, T. R., M. A. Toleman, L. Poirel, and P. Nordmann Metallo-β- lactamases: the quiet before the storm? Clin. Microbiol. Rev. 1: Weile, J., H. Rahmig, S. Gfröer, K. Schroeppel, C. Knabbe, and M. Susa First detection of a VIM-1 metallo-β-lactamase in a carbapenem-resistant Citrobacter freundii clinical isolate in an acute hospital in Germany. Scand. J. Infect. Dis. 39:-. 9

10 Table 1. Antibiotic susceptibilities of wild type and transformant strains. Antibiotics MIC (µg/ml) for P. aeruginosa E. coli DH5α 9551 (VIM-15) 1301 (VIM-1) VIM-15 VIM-1 pbc- pbc- pbc- VIM- Amoxicillin 51 > Host strain Piperacillin/ Tazobactam a Ceftazidime Cefotaxime >5 > Cefepime Aztreonam Meropenem 1 > Imipenem >1 > Gentamicin 0.5 > Tobramycin > a Tazobactam was used at a fixed concentration of µg/ml.

11 Table. Kinetic parameters of VIM-15, VIM-1, and VIM-. Substrate (s -1 ) Relative a VIM-15 VIM-1 VIM- K m /K m Relative a (µm) (s -1 µm -1 ) /K m (s -1 ) Relative a K m (µm) /K m (s -1 µm -1 ) Relative a /K m (s -1 ) Relative a K m /K m Relative a (µm) (s -1 µm -1 ) /K m Cephaloridine 190 ± 0 3 ± ± 0 0 ± ± ± Benzylpenicillin 0 ± ± ± 0 50 ± ± ± Cefepime 9.5 ± ± ± ± ± ± Ceftazidime 1.0 ± ± ± ± ± ± Cefotaxime 90 ± ± ± ± nd c nd nd nd nd Imipenem 1 ± ± ± 51 9 ± ± ± Meropenem.5 ± ± ± ± ± ± Aztreonam b ND d ND ND ND ND ND nd nd nd nd nd a The cephaloridine value was taken as 0%. b Hydrolysis of aztreonam was very slow; V max was estimated as times the maximum hydrolysis rate observed. c nd, not done d ND, Not determined. Hydrolysis was too slow to determine K m. 11