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

Laurent Poirela, Gerhard F. Weldhagenb, Christophe De Champsc and Patrice Nordmanna,*

a Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre; c Laboratoire de Bacteriologie, Faculté de Médecine, Université d'Auvergne, 63001 Clermont-Ferrand, France; b Department of Medical Microbiology, Faculty of Medicine, University of Pretoria, 0001 Pretoria, South Africa


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 Acknowledgements
 References
 
Eight Pseudomonas aeruginosa clinical strains that produce the clavulanic-acid-inhibited ß-lactamase GES-2 were isolated from patients of a South African hospital from March to July 2000. 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 ß-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 ß-lactamase.


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 Acknowledgements
 References
 
Clavulanic acid-inhibited expanded-spectrum ß-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-18.1–3 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 acid-inhibited 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 ß-lactamase gene among nosocomial P. aeruginosa strains in a South African hospital.


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 Acknowledgements
 References
 
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 ß-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 settings,5 the blaGES-1 primers (GES-1A and GES-1B5) were used to screen for blaGES-type genes. Using PCR and primers targeted to the sequences flanking blaGES-2 (GESCASA, 5'-ACAAAGATAATTTCCATCTCAAGGGAT-3'; GES-CASB, 5'-GTTTTAGACGGGCGTCAACT-3'),5 together with genomic DNA from the P. aeruginosa isolates, blaGES-2 was amplified. The PCR products were sequenced twice on both strands and analysed as reported previously.5

Since blaGES-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 blaGES-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 3.5–9.5 ampholine-containing polyacrylamide gel and ß-lactamase-containing extracts of overnight cultures.5 The pI values were determined by comparison with those of known ß-lactamases, including GES-2 ß-lactamase.

Nucleotide sequence accession number

The nucleotide sequences reported in this work have been assigned to the GenBank nucleotide database under accession number AF347174.


    Results and discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 Acknowledgements
 References
 
Epidemiology and PCR detection of ß-lactamase genes

Nine non-repetitive ceftazidime-resistant P. aeruginosa isolates were collected from nine hospitalized patients in Pretoria from March to July 2000. Of these nine isolates, eight were found to be blaGES 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 1Go). 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.


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Table 1. Clinical features of the ceftazidime-resistant blaGES-2 PCR-positive P. aeruginosa isolates
 
The infected patients were all individually at risk of acquiring P. aeruginosa (Table 1Go). Preliminary antibiotic susceptibility testing by disk diffusion assay showed that the P. aeruginosa isolates were resistant to most ß-lactams, all tested aminoglycosides and fluoroquinolones. These findings offer an explanation for the treatment failures involving ciprofloxacin or amikacin therapy (Table 1Go). 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 blaGES PCR-positive isolates with a mating-out frequency of 10-3–10-4.

MICs of ß-lactams for transconjugants mirrored those of P. aeruginosa clinical isolates (Table 2Go). The GES-2- producing P. aeruginosa strains were resistant to carbenicillins, ureidopenicillins, cefotaxime and ceftazidime and had reduced susceptibility to aztreonam (Table 2Go). The MIC of imipenem was eight-fold higher for the P. aeruginosa PU21 transconjugants than for P. aeruginosa PU21 (Table 2Go).


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Table 2. MICs of ß-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
 
IEF analysis of cell extracts of eight P. aeruginosa isolates and a single transconjugant revealed ß-lactamases with pI values of 5.8, 7.5 and 8–8.5. It was thought likely that the first, pI 5.8, was GES-2 and that the pI 8–8.5 was AmpC-type chromosome-encoded cephalosporinase.

Characterization of ß-lactamase genes and their genetic environment

In all cases, the same blaGES-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 blaGES-2, is linked to the same 59 base element recombination site found with the blaGES-2-like gene, blaIBC-1.8 The second gene cassette, located downstream of the ges-2 cassette, corresponds to an oxacillinase gene, blaOXA-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 ß-lactamases in the 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 ß-lactam resistance profile, weakly antagonized by clavulanate (Table 2Go), 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 blaVEB-1-positive P. aeruginosa isolates.3,4 Like the blaVEB-1-containing integrons, the blaGES-2-containing integrons code for two unrelated ß-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 ß-lactamases, respectively.6

This report illustrates that an outbreak of P. aeruginosa with a ß-lactamase-mediated resistance to imipenem may be due not only to a strain producing a class B metallo-enzyme (e.g. IMP-like ß-lactamase)1 but also, in part, to one producing a class A ß-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
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 Acknowledgements
 References
 
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.


    Notes
 
* Corresponding author. Tel: +33-1-45-21-36-32; Fax: +33-1-45-21-63-40; E-mail: nordmann.patrice{at}bct.ap-hop-paris.fr Back


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 Acknowledgements
 References
 
1 . Nordmann, P. & Guibert, M. (1998). Extended-spectrum ß-lactamases in Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy 42, 128–31.[Free Full Text]

2 . Naas, T., Philippon, L., Poirel, L., Ronco, E. & Nordmann, P. (1999). An SHV-derived extended-spectrum ß-lactamase in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 43, 1281–4.[Abstract/Free Full Text]

3 . Naas, T., Poirel, L., Karim, A. & Nordmann, P. (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.[ISI][Medline]

4 . Poirel, L., Rotimi, V. O., Mokaddas, E. M., Karim, A. & Nordmann, P. (2001). VEB-1 like extended-spectrum ß-lactamases in Pseudomonas aeruginosa from Kuwait. Emerging Infectious Diseases 7, 468–70.[ISI][Medline]

5 . Poirel, L., Weldhagen, G. F., De Champs, C., Naas, T., Dove, M. G. & Nordmann, P. (2001). Characterization of the class A ß-lactamase GES-2 from Pseudomonas aeruginosa with increased hydrolysis of imipenem. Antimicrobial Agents and Chemotherapy 45, 2598–603.[Abstract/Free Full Text]

6 . Girlich, D., Poirel, L., Leelaporn, A., Karim, A., Tribuddharat, C., Fennewald, M. 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]

7 . Campbell, M., Mahenthiralingam, E. & Speert, D. P. (2000). Evaluation of random amplified polymorphic DNA typing of Pseudomonas aeruginosa. Journal of Clinical Microbiology 38, 4614–5.[Abstract/Free Full Text]

8 . Giakkoupi, P., Tzouvelekis, L. S., Tsakris, A., Loukova, V., Sofianou, D. & Tzelepi, E. (2000). IBC-1, a novel integron-associated class A ß-lactamase with extended-spectrum properties produced by an Enterobacter cloacae clinical strain. Antimicrobial Agents and Chemotherapy 44, 2247–53.[Abstract/Free Full Text]

9 . Couture, F., Lachapelle, J. & Lévesque, R. C. (1992). Phylogeny of LCR-1 and OXA-5 with class A and class D beta-lactamases. Molecular Microbiology 6, 1693–705.[ISI][Medline]

10 . 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, 333–46.[Abstract]

Received 11 July 2001; returned 19 October 2001; revised 19 November 2001; accepted 5 December 2001