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Brazilian Journal of Infectious Diseases

Print version ISSN 1413-8670On-line version ISSN 1678-4391

Braz J Infect Dis vol.10 no.4 Salvador Aug. 2006

http://dx.doi.org/10.1590/S1413-86702006000400007 

ORIGINAL PAPERS

 

blaGES carrying Pseudomonas aeruginosa isolates from a public hospital in Rio de Janeiro, Brazil

 

 

Flávia L. P. C. PellegrinoI; Kátia R. Netto-dos SantosI; Lee W. RileyII; Beatriz M. Moreira I

IInstitute of Microbiology Professor Paulo de Góes, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil
IIInfectious Diseases Section, School of Public Health, Berkeley, California, USA

Address for correspondence

 

 


ABSTRACT

Previous analysis of Pseudomonas aeruginosa class-1 integrons from Rio de Janeiro, Brazil, revealed the blaGES gene in one isolate. We screened isolates of two widespread PFGE genotypes, A and B, at a public hospital in Rio, for the presence of blaGES. The gene was detected in all seven P. aeruginosa isolates belonging to genotype B. Three of the seven genotype-B isolates were resistant to amikacin, aztreonam, ceftazidime, cefepime, ciprofloxacin, gentamicin, imipenem, meropenem, piperacillin-tazobactam and ticarcillin-clavulanic acid. The other four isolates were resistant to all these agents, except gentamicin, imipenem, meropenem and piperacillin-tazobactam. A synergistic effect between ceftazidime and imipenem or clavulanic acid suggested the production of GES-type ESBL.

Key Words: Pseudomonas aeruginosa, blaGES, antibiotics, public hospital, Rio de Janeiro, Brazil.


 

 

Extended spectrum beta-lactamases (ESBLs) are enzymes with the ability to inactivate extended-spectrum cephalosporins and monobactams [1]. Various ESBL-types have been found in Pseudomonas aeruginosa, but new types are still emerging, including an extended spectrum beta-lactamase, GES [2]. Clinical laboratory detection of ESBL producing P. aeruginosa is important, because ESBL may confer resistance to ceftazidime (CAZ), an antimicrobial agent widely prescribed for P. aeruginosa infections [3].

GES-1 beta-lactamase was first detected in a Klebsiella pneumoniae isolate obtained in France in 1998 [2] from a child transferred from Cayenne, French Guiana. The gene, blaGES-1, conferred an extended-spectrum cephalosporin resistance profile, including clavulanic acid (CA), tazobactam and imipenem (IPM) [2]. The blaGES-1 gene was subsequently detected in P. aeruginosa from France, [4] and in K. pneumoniae from Portugal [5], isolated between 1999 and 2001. A GES-1 producing P. aeruginosa isolate was also detected in São Paulo, Brazil in the SENTRY surveillance program [6]. A new variant, GES-2, was described in P. aeruginosa from South Africa, isolated in 2000 [7], with the ability to confer intermediate resistance to IPM. Two other ESBLs, IBC-1 and IBC-2, were included in the group of GES-type beta-lactamases due similarity in the amino acid sequences. IBC-1 was described in an Enterobacter cloacae clinical isolate and IBC-2 in a P. aeruginosa clinical isolate, both obtained in Greece between 1998 and 2000 [8-10]. A double-disk synergy test with CAZ and IPM was able to identify 16 of 19 CAZ-resistant, blaIBC-1-carrying E. cloacae isolates [9]. The blaGES and blaIBC genes were found as gene cassettes integrated into class-1 integrons. Two other types of the GES enzyme were described (GES-3 and GES-4) in Klebsiella pneumoniae isolates from Japan [11, 12], also encoded by gene cassettes inserted into class-1 integrons located in plasmids.

Between 1999 and 2000, we detected two multidrug-resistant pulsed-field gel electrophoresis (PFGE) P. aeruginosa genotypes, named A and B, among 115 clinical isolates obtained at Hospital Universitário Clementino Fraga Filho (HUCFF), a public teaching hospital in Rio de Janeiro city, Brazil [13]. We subsequently detected the SPM metallo-beta-lactamase gene, and we studied class-1 integrons in these isolates (submitted for publication). The analyses of class-1 integrons revealed the blaGES gene in one genotype B isolate with a sequence identical to that reported for blaGES-1 (GenBank accession number AF355189). We screened all isolates belonging to genotype A and B from HUCFF for the presence of the blaGES gene and evaluated the detection of synergistic effects between CAZ and IPM or CA as a screening test for GES production.

 

Material and Methods

Bacterial isolates

All P. aeruginosa genotype A and B isolates from HUCFF were included in our study. Antimicrobial susceptibility of the 25 isolates belonging to genotype A (18) and B (7), based on disk-diffusion [14], revealed universal susceptibility only to polymyxin. Resistance rates to other antimicrobial agents were: 100% (CAZ, cefepime, ciprofloxacin and ticarcillin-CA), 96% (gentamicin, meropenem and piperacillin-tazobactam), 92% (amikacin and IPM) and 76% (aztreonam). Three of the seven genotype-B isolates were resistant to all the antimicrobial agents tested. The four other isolates were resistant to all of the agents, except gentamicin, IPM, meropenem and piperacillin-tazobactam.

Evaluation of antimicrobial synergy

A phenotypic test to evaluate possible synergistic effects with CAZ was performed by the addition of IPM (0.1µg and 1µg) or CA (10 µg) to CAZ (30µg)-containing disks placed on Mueller-Hinton agar medium inoculated with 0.5 McFarland density standard bacterial suspensions. The amount of IPM used in this test was previously determined to be sub-inhibitory for the growth of P. aeruginosa ATCC 27853. An enhancement of the CAZ inhibition zone (around 5 mm or more) in the presence of IPM or CA was interpreted as presumptive evidence for GES.

PCR screening for blaGES beta-lactamase

Chromosomal DNA was obtained from bacterial suspensions grown overnight in Luria Bertani broth with shaking, suspended in 100 mL of sterile water, and boiled for 10 min. After boiling the bacterial cells, PCR was performed on total DNA using GES-1A and GES-1B primers for the blaGES-1 gene [2] to screen all the isolates. The cycling parameters were 94ºC for 2 min, followed by 30 cycles of denaturation at 94ºC for 1 min, annealing at 50ºC for 1 min and extension at 72ºC for 1 min, with a final 4 min extension step at 72ºC. PCR products were visualized by electrophoresis at 90V for 2 h in 1.5% agarose gel stained with 1% ethidium bromide, visualized by UV transillumination, and photographed.

 

Results and Discussion

All genotype B isolates were positive for the blaGES gene by PCR, while all genotype A isolates were negative. A synergistic effect between CAZ and IPM was observed for four of the seven genotype-B isolates, tested by the addition of 0.1µg IPM to CAZ-containing disks (Figure 1). No synergistic effect was observed after the addition of 1µg IPM to CAZ disks. The test for synergy between CAZ and CA was positive for only three genotype-B isolates, including two with positive synergy to CAZ and IPM (Table 1). Genotype A isolates did not show any positive synergism.

 

 

 

 

GES is another example of a class-A ESBL in P. aeruginosa. The production of GES beta-lactamase has been associated with extended-spectrum cephalosporin resistance antagonized by the addition of various beta-lactams, including IPM [2, 9]. We found a positive synergism between CAZ and IPM for the blaGES gene-carrying isolates, reinforcing the utility of synergy tests to detect ESBL in P. aeruginosa. However, difficulties for this detection stem from several factors: false-negative results due to naturally-occurring beta-lactamases, such as over-expressed AmpC, the simultaneous presence of metallo-beta-lactamases or oxacillinases, relative resistance to inhibition by clavulanate, and combined resistance mechanisms, such as impermeability and efflux [15]. This is the first report describing a test for synergism in P. aeruginosa by addition of beta-lactamase inhibitors to cephalosporin-containing disks, as opposed to disk approximation or minimal inhibitory concentration (MIC) determination. This method could have an advantage over double-disk tests since interpretation of results appears to be easier.

GES-1 and IBC-1 are inhibited by IPM at relatively low concentrations, suggesting high affinity for the antibiotic [2, 9]. However, we only detected a synergistic effect when 0.1µg IPM was added to CAZ-containing disks. The addition of 1µg IPM to CAZ disks possibly induced AmpC-type beta-lactamase production by the P. aeruginosa isolate, which could have masked the inhibitory effect upon GES-1 ESBL. Consequently, the synergy tests may be useful as an initial screening for blaGES-1 producing P. aeruginosa isolates; but their efficiency still needs to be determined in order to substitute PCR methods to identify GES-type ESBL-producing isolates. Nucleotide sequence analysis of PCR products is still the only acceptable way to accurately discriminate between ESBL genes of the same family [15].

All seven genotype-B isolates from Rio carried the blaGES-1 gene, and four of these isolates revealed a synergistic effect between IPM and CAZ, evidenced by an increase of at least five mm in the inhibition zone obtained with a 30 µg CAZ disk, compared to that obtained with a 30 µg CAZ disk with 0.1 µg IPM added. Genotype B was the second-most-prevalent genotype detected in HUCFF in 1999-2000, showing a multidrug-resistance profile, including extended-spectrum cephalosporins and carbapenems. We found that genotype B isolates produced a gene coding an emerging ESBL. The identification of the blaGES gene in P. aeruginosa isolates from Brazil demonstrates that a new type of beta-lactamase has emerged on three continents within only four years. Rio de Janeiro city and São Paulo are located at least 3,000 km from the other places locations. Whether this gene was introduced into Brazil or if it appeared as a result of independent selection by antibiotic pressure within Brazil is yet to be determined. These observations are important, since confronting P. aeruginosa with multiple resistance mechanisms is becoming a challenge.

 

Acknowledgements

This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) of Brazil, and Fogarty International Program in Research and Training in Emerging Infectious Diseases (TW006563) of the National Institute of Health in the United States.

 

References

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13. Pellegrino F.L.P.C., Teixeira L.M., Carvalho M.G.S., et al. Occurrence of a multidrug-resistant Pseudomonas aeruginosa genotype in different hospitals in Rio de Janeiro, Brazil. J Clin Microbiol 2002;40:2420-24.         [ Links ]

14. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk susceptibility tests; Seventh edition. Approved standard M2-A7. NCCLS, Wayne, PA, 2000.         [ Links ]

15. Weldhagen G.F., Poirel L., Nordmann P. Ambler class A extended-spectrum beta-lactamases in Pseudomonas aeruginosa: Novel developments and clinical impact. Antimicrob Agents Chemother 2003;47:2385-92.        [ Links ]

 

 

Address for correspondence:
Dr. Beatriz Meurer Moreira, MD
Instituto de Microbiologia Professor Paulo de Góes, Bloco I, Centro de Ciências da Saúde, Cidade Universitária
21941-590, Rio de Janeiro, RJ, Brazil
Phone: 55-21-22604193, Fax. 55-21-25608344
E-mail: bmeurer@micro.ufrj.br.

Received on 13 February 2006; revised 16 June 2006.

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