JAC A nosocomial outbreak of Pseudomonas aeruginosa isolates expressing the extended-spectrum β-lactamase GES-2 in South Africa

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1 Journal of Antimicrobial Chemotherapy (2002) 49, JAC A nosocomial outbreak of Pseudomonas aeruginosa isolates expressing the extended-spectrum β-lactamase GES-2 in South Africa Laurent Poirel a, Gerhard F. Weldhagen b, Christophe De Champs c and Patrice Nordmann a * a Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Le Kremlin-Bicêtre; c Laboratoire de Bacteriologie, Faculté de Médecine, Université d Auvergne, Clermont-Ferrand, France; b Department of Medical Microbiology, Faculty of Medicine, University of Pretoria, 0001 Pretoria, South Africa Eight Pseudomonas aeruginosa clinical strains that produce the clavulanic-acid-inhibited β-lactamase GES-2 were isolated from patients of a South African hospital from March to July They were clonally related and each harboured a 150 kb conjugative plasmid carrying a class 1 integron containing a gene cassette encoding GES-2, followed by those for β-lactamase OXA-5 and an aminoglycoside modifying AAC(3)I-like enzyme. Hence, incidences of infection, several fatal, due to bacteria displaying clavulanate-inhibited resistance to extended-spectrum cephalosporins and reduced susceptibility to imipenem in Pretoria Academic Hospital, South Africa, can be explained, at least in part, by the spread of P. aeruginosa expressing the GES-2 β-lactamase. Introduction Clavulanic acid-inhibited expanded-spectrum β-lactamases (ESBLs) conferring resistance to extended-spectrum cephalosporins have been reported in Pseudomonas aeruginosa. 1 The clavulanic acid-inhibited enzymes that have been characterized in this species are the Ambler class A enzymes SHV2a, TEM-4, TEM-24, TEM-42, VEB-1-like enzymes and PER-1 and the class D oxacillinase OXA ESBL-producing P. aeruginosa have been reported from rare isolates in Europe, Kuwait and South East Asia, whereas PER-1 is widespread in Turkey. 1 4 We have recently described a novel clavulanic acidinhibited class A enzyme, GES-2, from a P. aeruginosa isolate from South Africa. 5 This enzyme is peculiar because it hydrolyses expanded-spectrum cephalosporins and, to a lesser extent, imipenem. The goal of this study was to analyse the spread of the GES-2 β-lactamase gene among nosocomial P. aeruginosa strains in a South African hospital. Material and methods Bacterial strains Between March 2000 and July 2000, a total of 361 consecutive P. aeruginosa isolates were collected, from different patients, by the Medical Microbiology department of the Pretoria Academic Hospital, South Africa. Of these 361 isolates, 72 were resistant to ceftazidime according to the results of preliminary susceptibility testing by disk diffusion. Of these, nine ceftazidime-resistant P. aeruginosa isolates, one per patient, were retained for further study. A rifampin-resistant mutant of P. aeruginosa PU21 was used as host in conjugation experiments. 5 Escherichia coli NCTC 50192, harbouring 154, 66, 38 and 7 kb plasmids, was used as the plasmid-containing reference strain. 5 Susceptibility testing and screening for ESBL-producing isolates The antibiotic susceptibilities of P. aeruginosa clinical isolates and E. coli transformants were first determined by disk diffusion test, 5 and the MICs of β-lactams were then determined as reported previously. 5 The double-disk synergy test was carried out on Mueller Hinton agar with disks containing ceftazidime and co-amoxiclav placed 2 cm apart. The results were interpreted as described previously. 5 PCR detection, cloning, sequencing and RAPD fingerprinting Using genomic DNA from ceftazidime-resistant P. aeruginosa isolates as templates and standard PCR amplification *Corresponding author. Tel: ; Fax: ; nordmann.patrice@bct.ap-hop-paris.fr 2002 The British Society for Antimicrobial Chemotherapy 561

2 L. Poirel et al. settings, 5 the bla GES-1 primers (GES-1A and GES-1B 5 ) were used to screen for bla GES -type genes. Using PCR and primers targeted to the sequences flanking bla GES-2 (GESCASA, 5 -ACAAAGATAATTTCCATCTCAAG- GGAT-3 ; GES-CASB, 5 -GTTTTAGACGGGCGTCA- ACT-3 ), 5 together with genomic DNA from the P. aeruginosa isolates, bla GES-2 was amplified. The PCR products were sequenced twice on both strands and analysed as reported previously. 5 Since bla GES-2 is accommodated on a gene cassette in a class 1 integron, 5 integron structures were studied using primers that hybridize to the 5 and 3 conserved segments (5 -CS and 3 -CS) of class 1 integrons. 5 Plasmid DNA from a P. aeruginosa PU21 transconjugant was partially digested with Sau3AI restriction endonuclease and cloned into BamHI-restricted pbk-cmv phagemid. Recombinant plasmids were selected in E. coli DH10B on TS agar containing 100 mg/l amoxicillin and analysed as reported previously. 6 Random amplified polymorphic DNA (RAPD) analysis was carried out with the selected P. aeruginosa isolates and interpreted as described previously. 6 The RAPD primers were ERIC-2, 628, AP1 and AP4. 6, 7 Conjugation, plasmid DNA content and IEF analyses Transfer of the ticarcillin resistance marker from bla GES - positive P. aeruginosa isolates to rifampin-resistant P. aeruginosa PU21 was attempted and plasmid DNA extractions were realized as reported previously. 5 Isoelectric focusing (IEF) analysis was carried out using ph ampholinecontaining polyacrylamide gel and β-lactamase-containing extracts of overnight cultures. 5 The pi values were determined by comparison with those of known β-lactamases, including GES-2 β-lactamase. Nucleotide sequence accession number The nucleotide sequences reported in this work have been assigned to the GenBank nucleotide database under accession number AF Results and discussion Epidemiology and PCR detection of β-lactamase genes Nine non-repetitive ceftazidime-resistant P. aeruginosa isolates were collected from nine hospitalized patients in Pretoria from March to July Of these nine isolates, eight were found to be bla GES positive (data not shown). Several P. aeruginosa isolates from different clinical specimens from each patient gave identical antibiotic resistance patterns according to antibiotic susceptibility testing by disk diffusion. The infected patients had been hospitalized in three different blocks of the hospital that are separated from one another by 100 m. One block contains the internal medicine ICU, the second the general surgery ICU and the third the neurosurgery ICU and the gynaecology ward (Table 1). Only patient 4 was hospitalized in the surgical ICU before moving to the internal medicine ICU. Since doctors and nurses move freely between these wards, the microbe could have been spread by medical staff, by undetected colonized and/or infected patients or by materials. The infected patients were all individually at risk of acquiring P. aeruginosa (Table 1). Preliminary antibiotic susceptibility testing by disk diffusion assay showed that the P. aeruginosa isolates were resistant to most β-lactams, all tested aminoglycosides and fluoroquinolones. These findings offer an explanation for the treatment failures involving ciprofloxacin or amikacin therapy (Table 1). No synergy between disks containing ceftazidime and clavulanate was found for the eight P. aeruginosa isolates. Genotypic comparison, plasmid analysis and transfer, susceptibility testing and IEF analysis Using RAPD analysis, the eight isolates were genotyped and found to be indistinguishable from each other (data not shown). An identical c. 150 kb conjugative plasmid was identified (data not shown) in transconjugants from the eight bla GES PCR-positive isolates with a mating-out frequency of MICs of β-lactams for transconjugants mirrored those of P. aeruginosa clinical isolates (Table 2). The GES-2- producing P. aeruginosa strains were resistant to carbenicillins, ureidopenicillins, cefotaxime and ceftazidime and had reduced susceptibility to aztreonam (Table 2). The MIC of imipenem was eight-fold higher for the P. aeruginosa PU21 transconjugants than for P. aeruginosa PU21 (Table 2). IEF analysis of cell extracts of eight P. aeruginosa isolates and a single transconjugant revealed β-lactamases with pi values of 5.8, 7.5 and It was thought likely that the first, pi 5.8, was GES-2 and that the pi was AmpC-type chromosome-encoded cephalosporinase. Characterization of β-lactamase genes and their genetic environment In all cases, the same bla GES-2 gene was identified by PCR followed by sequence analysis. Analysis of the nucleotide sequence of the 3369 bp insert of one recombinant plasmid, plap-2, revealed three open reading frames (ORFs). The first ORF, encoding bla GES-2, is linked to the same 59 base element recombination site found with the bla GES-2 -like gene, bla IBC-1. 8 The second gene cassette, located downstream of the ges-2 cassette, corresponds to an oxacillinase gene, bla OXA-5, associated with a 108 bp 59 base element. 9 This 59 base element sequence differs by six nucleotide subsitutions from that reported on the oxa-5 cassette. 9 The pi value of 7.5 found for one of the β-lactamases in the 562

3 Outbreak of GES-2-positive P. aeruginosa strains Table 1. Clinical features of the ceftazidime-resistant bla GES-2 PCR-positive P. aeruginosa isolates Patient Date of Date of Hospitalization Source of Other sources Underlying disease Treatment Outcome first isolation hospitalization unit first isolation 1 20 Mar 8 Mar internal medicine endotracheal urine chronic bronchitis/ ciprofloxacin imipenem improved ICU aspirate quadriplegia 2 21 Mar 14 Mar general surgical blood culture endotracheal multitrauma/sepsis imipenem amikacin died ICU aspirate 3 24 Mar 12 Mar general surgical endotracheal submandibular submandibular piperacillin tazobactam, amikacin died ICU aspirate tissue, urine infection/hiv 4 23 May 10 May internal medicine blood culture tracheostomy cerebral malaria/ aztreonam amikacin teicoplanin improved ICU entry site, sepsis/hiv endotracheal aspirate, arterial and catheter tips, urine 5 06 Jun 25 May neurosurgical endotracheal head trauma/sepsis ciprofloxacin amikacin died ICU aspirate 6 26 Jun 20 Jun neurosurgical endotracheal head trauma/sepsis ciprofloxacin amikacin died ICU aspirate 7 27 Jun 2 Jun neurosurgical endotracheal head trauma/sepsis/ ciprofloxacin amikacin died ICU aspirate subdural bleeding 8 7 Jul 28 Jun gynaecology ward pus swab wound sepsis after antiseptic (no systematic antibiotics) improved Caesarean section 563

4 L. Poirel et al. Table 2. MICs of β-lactams for P. aeruginosa clinical isolates, a P. aeruginosa PU21 transconjugant, E. coli DH10B harbouring recombinant plasmids plap-1, plap-2 and plap-3, and reference strains P. aeruginosa PU21 and E. coli DH10B MIC (mg/l) for: P. aeruginosa P. aeruginosa P. aeruginosa PU21 E. coli DH10B E. coli DH10B E. coli DH10B E. coli DH10B clinical isolates transconjugant (plap-1) b (plap-2) c (plap-3) d Antibiotic(s) a (GES-2/OXA-5) (GES-2/OXA-5) (GES-2) (GES-2/OXA-5) (OXA-5) Amoxicillin >512 >512 >512 >512 >512 >512 4 Amoxicillin CLA >512 >512 > Ticarcillin >512 > >512 4 Ticarcillin CLA > Ticarcillin TZB > Piperacillin Piperacillin CLA Piperacillin TZB Cefalothin >512 >512 >512 > Cefoxitin >512 >512 > Ceftazidime Ceftazidime CLA Ceftazidime TZB Cefotaxime Cefotaxime CLA Cefotaxime TZB Cefepime Aztreonam Imipenem Meropenem <0.06 <0.06 Moxalactam Sulphonamides e >256 1 e >256 > Kanamycin e >256 >512 e Gentamicin e 12 1 e a CLA, clavulanic acid at a fixed concentration of 2 mg/l; TZB, tazobactam at a fixed concentration of 4 mg/l. b Recombinant plasmid plap-1 was obtained previously. 5 c Recombinant plasmid plap-2 possesses the ges-2, oxa-5 and aac(3)i-like gene cassettes. d Recombinant plasmid plap-3 possesses the oxa-5 and aac(3)i-like gene cassettes. e Not done, since not relevant. 564

5 Outbreak of GES-2-positive P. aeruginosa strains P. aeruginosa clinical isolates, the PU21 transconjugant and the E. coli DH10B (plap-2) corresponds to that of OXA-5. 9 The third integron-located gene cassette accommodates an aac(3)i variant, encoding a 3-N-aminoglycoside acetyltransferase. 10 This aminoglycoside resistance modifying protein differs by five amino acids from that reported previously (Lys15Glu, Lys51Arg, Asp60Glu, Glu142Asp, Thr152Ser). 10 It conferred on E. coli DH10B (plap-2) resistance to kanamycin and gentamicin. This class 1 integron has a classical 3 -CS end that encodes a sulphonamide resistance determinant. A second recombinant plasmid, plap-3, that contained only the oxa-5 and aac(3)i cassettes was analysed. E. coli DH10B (plap-3) showed a narrow-spectrum β-lactam resistance profile, weakly antagonized by clavulanate (Table 2), consistent with production of a narrow-spectrum oxacillinase with a pi of 7.5. Unexpectedly, using 5 -CS and 3 -CS primers targeted to the ends of class 1 integrons, multiple DNA fragments were obtained, of sizes c. 2, 1.5 and 1 kb, for each P. aeruginosa clinical isolate and its transconjugant. The sizes and restriction patterns of these PCR fragments (data not shown) correspond to integrons with one, two or all three of the gene cassettes. Each has the GES-2 gene cassette, two have the oxa-5 cassette and one also has the aac(3)i cassette. The polymorphism detected by PCR probably reflects a single class 1 integron containing three antibiotic resistance genes and derivatives of it arising from excision of the oxa-5 and aac(3)i cassettes. Conclusion The work reported here describes an outbreak of P. aeruginosa harbouring a 150 kb conjugative plasmid containing a class 1 integron and expressing a class A ESBL with hydrolytic activity that encompasses imipenem. Plasmid and integron locations for ESBL genes have been found to date only in rare bla VEB-1 -positive P. aeruginosa isolates. 3,4 Like the bla VEB-1 -containing integrons, the bla GES-2 -containing integrons code for two unrelated β-lactamases, an Ambler class A ESBL and a narrow-spectrum class D enzyme. The oxacillinases OXA-10 and OXA-5 (82% amino acid identity) are associated with VEB-1 and now GES-2 β-lactamases, respectively. 6 This report illustrates that an outbreak of P. aeruginosa with a β-lactamase-mediated resistance to imipenem may be due not only to a strain producing a class B metalloenzyme (e.g. IMP-like β-lactamase) 1 but also, in part, to one producing a class A β-lactamase (GES-2). The combination of genes encoding clavulanate-resistant oxacillinases and class A ESBL genes, in the same isolates, may complicate detection of these multi-resistant isolates. Acknowledgements We thank Michael G. Dove for continuous support of G. F. Weldhagen s work. This work was funded by a grant from the Ministères de l Education Nationale et de la Recherche (UPRES, JE 2227), Université Paris XI, Faculté de Médecine Paris-Sud, France. References 1. Nordmann, P. & Guibert, M. (1998). Extended-spectrum β-lactamases in Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy 42, Naas, T., Philippon, L., Poirel, L., Ronco, E. & Nordmann, P. (1999). An SHV-derived extended-spectrum β-lactamase in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 43, Naas, T., Poirel, L., Karim, A. & Nordmann, P. (1999). Molecular characterization of In50, a class 1 integron encoding the gene for the extended-spectrum β-lactamase VEB-1 in Pseudomonas aeruginosa. FEMS Microbiology Letters 176, Poirel, L., Rotimi, V. O., Mokaddas, E. M., Karim, A. & Nordmann, P. (2001). VEB-1 like extended-spectrum β-lactamases in Pseudomonas aeruginosa from Kuwait. Emerging Infectious Diseases 7, Poirel, L., Weldhagen, G. F., De Champs, C., Naas, T., Dove, M. 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Phylogeny of LCR-1 and OXA-5 with class A and class D beta-lactamases. Molecular Microbiology 6, Javier Teran, F., Alvarez, M., Suarez, J. E. & Mendoza, J. C. (1991). Characterization of two aminoglycoside (3)-N-acetyltransferase genes and assay as epidemiological probes. Journal of Antimicrobial Chemotherapy 28, Received 11 July 2001; returned 19 October 2001; revised 19 November 2001; accepted 5 December

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