hydrophila strain ACCEPTED *Corresponding author. Department of Bacteriology, National Center for Epidemiology, 1097

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1 JCM Accepts, published online ahead of print on 26 March 2008 J. Clin. Microbiol. doi: /jcm Copyright 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved Identification of the first VIM metallo-β-lactamase producing multiresistant Aeromonas hydrophila strain Running title: VIM metallo-β-lactamase producing A. hydrophila Balázs Libisch a*, Christian G. Giske b, Bogáta Kovács a, Tamás G. Tóth c, Miklós Füzi a a Department of Bacteriology, National Center for Epidemiology, Budapest, Hungary; b Department of Clinical Microbiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; c Department of Internal Medicine, St. John's Hospital, Budapest, Hungary *Corresponding author. Department of Bacteriology, National Center for Epidemiology, 1097 Budapest, Gyáli út 2-6, Hungary. Phone: ; Fax.: libischb@oek.antsz.hu 1

2 Abstract A VIM metallo-β-lactamase producing Aeromonas hydrophila strain carrying an integron-borne bla VIM-4 gene was isolated from a cirrhotic patient s faecal sample in a Budapest hospital. The variable region of this integron is identical with that of a previously characterised integron from Pseudomonas aeruginosa clinical isolates in Pécs, South Hungary. Keywords: Metallo-β-lactamase, bla VIM, Aeromonas hydrophila, Acquired, Multidrug resistant 2

3 Aeromonas species are considered food-borne pathogens of emerging importance (2, 7, 15). Most species belonging to this genus, particularly those associated with human infections are widely distributed in the environment, especially in freshwater, sewage, marine environments, drinking water and are also found in a wide range of animal and plant food products (1, 2, 5, 21). These pathogens can cause infections both in immunocompetent and -compromised patients including gastroenteritis, bacteraemia, meningitis, skin and soft tissue infections (9). Chromosomally mediated, inducible β-lactamases were recognized as the major mechanism of β-lactam resistance in Aeromonas hydrophila. These enzymes comprise a molecular class C cephalosporinase, a class D penicillinase and a class B metallo-β-lactamase (MBL) designated CphA, the latter being a carbapenemase enzyme with a relatively narrow substrate profile (1, 16, 19, 20). In this report we describe the first VIM MBL producing A. hydrophila strain carrying an integron-borne bla VIM-4 gene. Antimicrobial disk susceptibility tests were performed on Mueller-Hinton agar (Oxoid, Basingstoke, UK) as recommended by the Clinical and Laboratory Standards Institute (6). Test disks were purchased from Oxoid. MICs were determined by the agar dilution method for β-lactam antibiotics and by the Etest (AB Biodisk, Solna, Sweden) for other antibiotics. To detect MBL production the MBL Etest (AB Biodisk, Solna, Sweden) and the Imipenem-EDTA Disk method (23) were used and complemented with the use of ceftazidime, cefepime, cefotaxime, cefoxitin, cefoperazone, piperacillin and piperacillin-tazobactam disks ± 750 µg EDTA. To prepare β- lactam/edta disks 750 µg EDTA in the form of a 0.5 M EDTA solution (ph 8.0) was added to the β-lactam disk placed on a Mueller-Hinton agar plate (3, 23). Detection of bla VIM genes and class 1 integrons by PCR and sequencing was performed as described earlier (13, 14). Conjugation experiments were performed on Mueller-Hinton agar plates with the E. coli J5-3 Rif R and the Pseudomonas aeruginosa PAO4089Rp strains (12) as recipients. 3

4 Transconjugants were selected on Mueller-Hinton agar plates containing 300 and 100 µg/ml rifampicin, respectively and 32 µg/ml cefotaxime or 128 µg/ml ticarcillin. The A. hydrophila isolate MB443 was recovered in October 2005 from the faecal sample of a 68- year-old male patient suffering from alcoholic liver disease (cirrhosis) associated with pathologic fluid accumulation within the abdominal cavity. According to the anamnesis recorded in the Budapest hospital the clinical history of the patient included childhood hepatitis and later appendectomy, but no relevant antibiotic treatment. After the drainage of the ascites fluid the patient was released from the hospital and started on a diuretic therapy. A. hydrophila was not isolated from any other sample types. The antimicrobial susceptibility profile of isolate MB443 is shown in Table 1 together with the corresponding values determined for the control A. hydrophila strain ATCC7966. While MB443 was resistant to ceftazidime, cefepime, piperacillin-tazobactam and imipenem, it remained susceptible to aztreonam. This profile is often observed among acquired MBL-producing gram-negative clinical isolates (14). Although Aeromonas spp. possess a chromosomal MBL that hydrolyzes the carbapenems, wild- type strains are usually categorized under standardized in vitro testing conditions as carbapenem susceptible (20, 22). Under selective pressure of certain antibiotics the emergence of β-lactamase overexpressing carbapenem-resistant Aeromonas spp. mutant strains has been observed (8, 10). The MBL Etest does not detect the presence of the chromosomal MBLs in wild-type strains (22). For isolate MB443 and for the control strain the MIC values of the MBL Etest could not be determined as the inhibitory ellipse did not reach the calibrated scale on the strip (Fig. 1). However, for isolate MB443 a 10 mm increase of the inhibitory zones was observed around all ß-lactam disks tested after the addition of 750 µg EDTA to the disks (Table 2). Moreover, a typical synergistic zone was 4

5 detected between the cefoxitin and the cefoxitin-edta disks (Fig. 2). These observations indicated the presence of an acquired MBL with a much broader substrate profile than CphA. PCR experiments detected a bla VIM-4 gene located on a class 1 integron. The variable region of this integron was sequenced and it was found to be identical with that of an integron previously characterised from P. aeruginosa isolates in Pécs, South Hungary with GenBank accession number AY (Fig. 3; ref. 14). This integron carries two resistance gene cassettes, an aaca4 gene in the first position followed by a bla VIM-4 gene cassette that contains a 170 bp duplication. To the best of our knowledge this is the first example of a VIM MBL producing isolate within the Aeromonas genus worldwide. Mating out assays failed to transfer the bla VIM gene to the recipient E. coli and P. aeruginosa strains under the experimental conditions applied. This observation is similar to several previous reports of conjugation experiments with VIM-producing clinical isolates as donors (13, 14). The potential of Aeromonas species carrying antibiotic resistance genes to enter the food chain and subsequently infect or colonise humans deserves attention. Class 1 integrons that harbour trimethoprim-sulfamethoxazole, tetracycline, aminoglycoside and chloramphenicol resistance determinants and also an IMP MBL were detected in Aeromonas spp. in previous studies (4, 5, 11, 17, 21). In this report we identified an integron borne bla VIM-4 gene from A. hydrophila, a previously unreported horizontally acquired resistance mechanism in this genus. The results of the MBL phenotypic tests may also have implications for the screening protocols. As the MBL Etest failed to detect the VIM enzyme in A. hydrophila, screening by the use of inhouse disks ± EDTA or by PCR could be a recommended strategy for the detection of acquired MBLs in Aeromonas spp. The origin of acquired MBLs of human pathogens is still unknown (18). Aeromonas spp. isolates carrying such genes could provide a link between their supposed environmental pool and human pathogenic bacteria. Based on our observations further molecular and metagenomic studies 5

6 are needed to identify and characterise integron-containing bacteria from environmental sources that share the same ecological niches with the motile aeromonads. This work was financially supported by the European Union through the DRESP2 FP6 grant with no. LSHM-CT References 1. Abbott, S. L Aeromonas. p In P. R. Murray, E. J. Baron, M. A. Pfaller, J. H. Jorgensen, and R. H. Yolken (ed.), Manual of clinical microbiology, 8 th ed., ASM Press, Washington D.C. 2. Adams, M. R., M. O. Moss Bacterial agents of foodborne illness. Aeromonas hydrophila. p In Food Microbiology. 2 nd ed., The Royal Society of Chemistry, Cambridge, UK. 3. Andrade, S. S., R. C. Picão, E. H. Campana, A. G. Nicoletti, A. C. Pignatari, A. C. Gales Influence of disk preparation on detection of metallo-β-lactamase-producing isolates by the combined disk assay. J. Clin. Microbiol. 45: Barlow, R. S., J. M. Pemberton, P. M. Desmarchelier, K. S. Gobius Isolation and characterization of integron-containing bacteria without antibiotic selection. Antimicrob. Agents Chemother. 48: Chang, Y. C., D. Y. Shih, J. Y. Wang, S. S. Yang Molecular characterization of class 1 integrons and antimicrobial resistance in Aeromonas strains from foodborne outbreaksuspect samples and environmental sources in Taiwan. Diagn. Microbiol. Infect. Dis. 59: Clinical and Laboratory Standards Institute Procedure for performing the disk diffusion test. p In Performance Standards for Antimicrobial Disk Susceptibility Tests. 8 th ed. M02-A8. Wayne, PA 6

7 Daskalow, H The importance of Aeromonas hydrophila in food safety. Food Control 17: Iaconis, J. P., C. C. Sanders Purification and characterization of inducible β- lactamases in Aeromonas spp. Antimicrob. Agents Chemother. 34: Jones, B. L., M. H. Wilcox Aeromonas infections and their treatment. J. Antimicrob. Chemother. 35: Ko, W. C., H. M. Wu, T. C. Chang, J. J. Yan, J. J. Wu Inducible β-lactam resistance in Aeromonas hydrophila: therapeutic challenge for antimicrobial therapy. J. Clin. Microbiol. 36: L'Abee-Lund, T. M., H. Sorum Class 1 integrons mediate antibiotic resistance in the fish pathogen Aeromonas salmonicida worldwide. Microb. Drug Resist. 7: Lee, K., J. B. Lim, J. H. Yum, D. Yong, Y. Chong, J. M. Kim, D. M. Livermore bla VIM-2 cassette-containing novel integrons in metallo-β-lactamase-producing Pseudomonas aeruginosa and Pseudomonas putida isolates disseminated in a Korean hospital. Antimicrob. Agents Chemother. 46: Libisch, B., M. Gacs, K. Csiszar, M. Muzslay, L. Rokusz, M. Fuzi Isolation of an integron-borne bla VIM-4 type metallo-β-lactamase gene from a carbapenem-resistant Pseudomonas aeruginosa clinical isolate in Hungary. Antimicrob. Agents Chemother. 48: Libisch, B., M. Muzslay, M. Gacs, J. Minarovits, M. Knausz, J. Watine, G. Ternak, E. Kenez, I. Kustos, L. Rokusz, K. Szeles, B. Balogh, M. Fuzi Molecular epidemiology of VIM-4 metallo-β-lactamase-producing Pseudomonas sp. isolates in Hungary. Antimicrob. Agents Chemother. 50:

8 Lindberg, M. R., J. M. Havens, G.Y. Lauwers, M. A. Scott, L. W. Lamps Aeromonas: An emerging food-borne cause of infectious colitis. Mod. Pathol. 21:127A. 16. Majiduddin, F. K., I. C. Materon, T. G. Palzkill Molecular analysis of β-lactamase structure and function. Int. J. Med. Microbiol. 292: Neuwirth C., E. Siebor, F. Robin, R. Bonnet First occurrence of an IMP metallo-β- lactamase in Aeromonas caviae: IMP-19 in an isolate from France. Antimicrob Agents Chemother. 51: Quinteira S., L. Peixe Multiniche screening reveals the clinically relevant metallo-β- lactamase VIM-2 in Pseudomonas aeruginosa far from the hospital setting: an ongoing dispersion process? Appl. Environ Microbiol. 72: Rasmussen, B. A., K. Bush. Carbapenem-hydrolyzing β-lactamases Antimicrob. Agents Chemother. 41: Rossolini, G. M., T. Walsh, G. Amicosante The Aeromonas metallo-β-lactamases: genetics, enzymology, and contribution to drug resistance. Microb. Drug Resist. 2: Schmidt, A. S., M. S. Bruun, I. Dalsgaard, J. L. Larsen Incidence, distribution, and spread of tetracycline resistance determinants and integron-associated antibiotic resistance genes among motile aeromonads from a fish farming environment. Appl. Environ Microbiol. 67: Walsh, T. R., A. Bolmstrom, A. Qwarnstrom, A. Gales Evaluation of a new Etest for detecting metallo-β-lactamases in routine clinical testing. J. Clin. Microbiol. 40:

9 Yong, D., K. Lee, J. H. Yum, H. B. Shin, G. M. Rossolini, Y. Chong Imipenem- EDTA disk method for differentiation of metallo-β-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J. Clin. Microbiol. 40:

10 FIGURE LEGENDS Fig. 1. Etest MBL strip for the A. hydrophila isolate MB443. IP stands for imipenem concentrations (4-256 µg/ml) and IPI for imipenem (1-64 µg/ml) plus a constant level of EDTA. IP and IPI MIC values should be read where the respective inhibition ellipses intersect the strip (AB Biodisk, Solna, Sweden). Fig. 2. Inhibitory zones for the A. hydrophila isolate MB443 around ß-lactam disks ± 750 µg EDTA. FOX, cefoxitin; CTX, cefotaxime; TZP, piperacillin-tazobactam. Fig. 3. The variable region of the integron of the VIM-4-producing A. hydrophila isolate MB443 characterised in this study. The empty ellipse represents the atti1 site, black circles the 59-base elements. The asterisk indicates a bla VIM-4 cassette with the 170 bp duplication. 5 -CS and 3 -CS stand for the 5 and 3 conserved sequences, respectively. 10

11 TABLE 1. Antibiotic susceptibilities for the A. hydrophila strains MB443 and ATCC7966 Strain MIC (µg/ml) a AMP TZP FOX CAZ CTX CRO FEP IPM ATM GEN AMK TOB CIP TET CHL POB MB443 >256 >256 >256 >256 >256 > < > ATCC7966 > <0.5 <0.5 <0.5 < < < <0.5 2 a AMP, ampicillin; TZP, piperacillin-tazobactam; FOX, cefoxitin; CAZ, ceftazidime; CTX, cefotaxime; CRO, ceftriaxone, FEP, cefepime; IPM, imipenem; ATM, aztreonam; GEN, gentamicin; AMK, amikacin; TOB, tobramycin; CIP, ciprofloxacin; TET, tetracycline; CHL, chloramphenicol; POB, polymyxin B 11

12 TABLE 2. Diameter values of inhibitory zones measured in the absence and presence of EDTA Inhibitory zone (mm) Antibiotic Disk (µg) MB443 ATCC7966 EDTA + EDTA EDTA + EDTA Imipenem Ceftazidime Cefepime Cefotaxime Cefoxitin Cefoperazone Piperacillin Piperacillin- 100/ tazobactam 12

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15 5 -CS aaca4 bla VIM-4 3 -CS