Antimicrobial Agents and Chemotherapy, June 2004, p . 2277-2279, Vol . 48, No . 6

Nosocomial Outbreak of Extended-Spectrum ß-Lactamase SHV-5-Producing Isolates of Pseudomonas aeruginosa in Athens, Greece

Laurent Poirel,¹ Evangelia Lebessi,² Marisa Castro,¹ Cindy Fèvre,¹ Maria Foustoukou,² and Patrice Nordmann^1*

Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre, France,¹ Department of Clinical Microbiology, P . and A . Kyriakou Children's Hospital, Athens, Greece²

Received 22 August 2003/ Returned for modification 16 November 2003/ Accepted 21 February 2004

The ESBL SHV-5 was reported first in a Klebsiella pneumoniae isolate from Chile in 1989 (6) . Subsequently, the bla_SHV-5 gene was identified in several enterobacterial species scattered throughout the world and especially in Greece (3, 7, 17) . The ß-lactamase SHV-5 confers a high level of resistance to ceftazidime and to monobactams .

The aim of our study was to analyze the spread of P . aeruginosa isolates expressing an ESBL phenotype and recovered in different units of a pediatric hospital in Athens, Greece, from 1998 to 2002 . Between July 1998 and February 2002, 50 consecutive ceftazidime-resistant P . aeruginosa strains identified by using the API32GN system (bioMérieux, Marcy-l'Etoile, France) were isolated from different patients in the clinical microbiology laboratory of P . and A . Kyriakou Children's Hospital in Athens, Greece . Among these strains, seven isolates had an ESBL phenotype according to results of a double-disk synergy test performed with cefepime, ceftazidime, and ticarcillin-clavulanic acid disks on Mueller-Hinton agar plates, as described previously (10) . P . aeruginosa isolates 1 to 7 were from hospitalized patients, and their clinical backgrounds are detailed in Table 1 . Four out of the seven patients were colonized by the ESBL-positive P . aeruginosa isolates, whereas three of the patients had infections and received antibiotic regimens consisting of piperacillin-tazobactam (two patients) or meropenem (one patient) (Table 1) . Isolates 1 to 6 were resistant to ceftazidime, cefotaxime, cefpirome, cefepime, and aztreonam . They were susceptible to ticarcillin-clavulanate, piperacillin-tazobactam, imipenem, and meropenem (Table 2) . Isolate 7 exhibited a higher level of resistance for most ß-lactams . For all the strains, MICs of ceftazidime and cefotaxime were lowered by addition of clavulanate, which was consistent with ESBL expression (Table 2) . In addition, all isolates were resistant to fluoroquinolones, gentamicin, amikacin, and tobramycin except for isolate 7, which remained susceptible to fosfomycin and ciprofloxacin . All isolates were susceptible to colistin, according to the guidelines of Gales et al . (2) using an agar dilution method .

 
PCR experiments using primers specific for bla_TEM, bla_SHV, bla_VEB-1, bla_GES-1, and bla_PER-1 (4) gave a positive result only for the bla_SHV gene . Sequence analysis of the entire gene revealed its perfect identity with the bla_SHV-5 gene .

Isoelectric focusing analysis performed as described elsewhere (14) showed that P . aeruginosa isolates expressed two ß-lactamases with pI values of 8.2 and 8.4 that likely corresponded to SHV-5 and the naturally occurring AmpC-type enzyme, respectively .

Transfer of the ceftazidime resistance marker of P . aeruginosa isolate 7 by conjugation or electroporation as described previously (13) using ciprofloxacin-resistant P . aeruginosa PU21 as the recipient strain failed . The location of the bla_SHV-5 gene was determined precisely by using the endonuclease I-CeuI technique (9) . Pulsed-field gel electrophoresis (PFGE) gave four DNA fragments from P . aeruginosa isolates 1 (0.5, 1.5, 1.7, and 2.2 Mb) and 7 (0.7, 1.3, 1.7, and 2.2 Mb) and also from the P . aeruginosa PU21 reference strain (0.5, 1.1, 1.9, and 2.2 Mb) (Fig . 1) . The DNA probe for rRNA consisting of a 1,504-bp PCR fragment for 16S and 23S rRNA genes (4) hybridized with all the fragments of all P . aeruginosa DNAs except those of 2.2 Mb (Fig . 1) . Hybridization with DNA probe internal to bla_SHV-5 consisting of a 300-bp PCR fragment generated from whole-cell DNA of P . aeruginosa gave a single signal for isolate 1, suggesting that bla_SHV-5 was chromosomally located (Fig . 1) . Hybridization with a DNA probe for bla_SHV-5 gave three signals for isolate 7, indicating that at least three copies of the bla_SHV-5 gene were scattered on the chromosome of that strain (Fig . 1) .

 
PCRs using primers annealing to bla_SHV-5 and the IS26 transposase gene did not provide positive results, whereas IS26 was found to be associated with bla_SHV-2a (11), and detection of the bla_SHV-5 gene as part of a class 1 integron by PCR also failed (8) .

The SHV-5-producing P . aeruginosa isolates were recovered in different units of the P . and A . Kyriakou Children's Hospital during a 5-year period . Thus, it was hypothesized that one clone may have persisted in that hospital . Consequently, genotypic comparison was performed in order to evaluate the clonality of the isolates . PFGE analysis performed with the restriction enzyme SpeI as described previously (5) showed that six out of the seven isolates likely corresponded to a single clone, whereas isolate 7 was not clonally related (Fig . 2) (15) .

 
The present work described ESBL-producing P . aeruginosa isolates in several hospitalization units of a pediatric hospital . ESBL production is frequently reported in Enterobacteriaceae but rarely in P . aeruginosa (18) . As previously observed (13), the infected patients were at risk of acquiring P . aeruginosa infections . Two distinct clonal isolates were found, demonstrating coexistence of distinct ESBL-positive clones in the same unit . Another threatening aspect was that the same P . aeruginosa clone had been involved in nosocomial infections during a 4-year period of time, suggesting probable persistence of the isolate in the hospital environment .

Interestingly, the presence in the chromosome of a single isolate of several copies of the same ß-lactamase gene may explain in part the higher level of resistance to ß-lactams of that strain compared to others . It may indicate a probable location of the ß-lactamase gene bla_SHV-5 on a transposable element, although this has not been identified .

This study represents the first description of the bla_SHV-5 gene in the Pseudomonas species and it adds to the list of ESBLs identified in P . aeruginosa. While this work was in progress, an outbreak of 11 P . aeruginosa isolates producing SHV-5 was reported in Heraklion, Crete (12), which is in the vicinity of Athens .

In our study, the bla_SHV-5 gene was chromosomally located and might have resulted from transfer of a bla_SHV-5-positive plasmid from Enterobacteriaceae followed by its chromosomal integration (7) . Finally, this study suggested that when ESBLs become very much prevalent in Enterobacteriaceae, they may become the source of acquired resistance in P . aeruginosa that may remain hidden if not systematically investigated .

1. Chen, H . Y., M . Yuan, and D . M . Livermore. 1995 . Mechanisms of resistance to ß-lactam antibiotics amongst Pseudomonas aeruginosa isolates collected in the UK in 1993 . J . Med . Microbiol . 43:300-309.
2. Gales, A . C., A . O . Reis, and R . N . Jones. 2001 . Contemporary assessment of antimicrobial susceptibility testing methods for polymyxin B and colistin: review of available interpretative criteria and quality control guidelines . J . Clin . Microbiol . 39:183-190.
3. Gianneli, D., E . Tzelepi, L . S . Tzouvelekis, A . F . Mentis, and C . Nikolopoulou. 1994 . Dissemination of cephalosporin-resistant Serratia marcescens strains producing a plasmidic SHV type ß-lactamase in Greek hospitals . Eur . J . Microbiol . Infect . Dis . 13:764-767.
4. Girlich, D., L . Poirel, A . Leelaporn, A . Karim, C . Tribuddharat, M . Fennewald, and P . Nordmann. 2001 . Molecular epidemiology of the integron-located VEB-1 extended-spectrum ß-lactamase in nosocomial enterobacterial isolates in Bangkok, Thailand . J . Clin . Microbiol . 39:175-182.
5. Girlich, D., T . Naas, A . Leelaporn, L . Poirel, M . Fennewald, and P . Nordmann. 2002 . Nosocomial spread of the integron-located veb-1-like cassette encoding an extended-spectrum ß-lactamase in Pseudomonas aeruginosa in Thailand . Clin . Infect . Dis . 34:175-182.
6. Gutmann, L., B . Ferré, F . W . Goldstein, N . Rizk, E . Pinto-Schuster, J . F . Acar, and E . Collatz. 1989 . SHV-5, a novel SHV-type ß-lactamase that hydrolyzes broad-spectrum cephalosporins and monobactams . Antimicrob . Agents Chemother . 33:951-956.
7. Legakis, N . J., L . S . Tzouvelekis, G . Hatzoudis, E . Tzelepi, A . Gourkou, T . L . Pitt, and A . C . Vatopoulos. 1995 . Klebsiella pneumoniae infections in Greek hospitals . Dissemination of plasmids encoding an SHV-5-type beta-lactamase . J . Hosp . Infect . 31:177-187.
8. Lévesque, C., L . Piché, C . Larose, and P . H . Roy. 1995 . PCR mapping of integrons reveals several novel combinations of resistance genes . Antimicrob . Agents Chemother . 39:185-191.
9. Liu, S . L., A . Hessel, and K . E . Sanderson. 1993 . Genomic mapping with I-Ceu I, an intron-encoded endonuclease specific for genes for ribosomal RNA, in Salmonella spp., Escherichia coli, and other bacteria . Proc . Natl . Acad . Sci . USA 90:6874-6878.
10. Luzzaro, F., E . Mantengoli, M . Perilli, G . Lombardi, V . Orlandi, A . Orsatti, G . Amicosante, G . M . Rossolini, and A . Toniolo. 2001 . Dynamics of a nosocomial outbreak of multidrug-resistant Pseudomonas aeruginosa producing the PER-1 extended-spectrum ß-lactamase . J . Clin . Microbiol . 39:1865-1870.
11. Naas, T., L . Philippon, L . Poirel, E . Ronco, and P . Nordmann. 1999 . An SHV-derived extended-spectrum ß-lactamase in Pseudomonas aeruginosa. Antimicrob . Agents Chemother . 43:1281-1284.
12. Neonakis, I . K., E . V . Scoulica, S . K . Dimitriou, A . I . Gikas, and Y . J . Tselentis. 2003 . Molecular epidemiology of extended-spectrum ß-lactamases produced by clinical isolates in a university hospital in Greece: detection of SHV-5 in Pseudomonas aeruginosa and prevalence of SHV-12 . Microb . Drug Resist . 9:161-165.
13. Poirel, L., G . F . Weldhagen, C . De Champs, and P . Nordmann. 2002 . A nosocomial outbreak of Pseudomonas aeruginosa isolates expressing the extended-spectrum ß-lactamase GES-2 in South Africa . J . Antimicrob . Chemother . 49:561-565.
14. Poirel, L., M . Guibert, S . Bellais, T . Naas, and P . Nordmann. 1999 . Integron- and carbenicillinase-mediated reduced susceptibility to amoxicillin-clavulanic acid in isolates of multidrug-resistant Salmonella enterica serotype Typhimurium DT104 from French patients . Antimicrob . Agents Chemother . 43:1098-1104.
15. Tenover, F . C., R . D . Arbeit, R . V . Goering, P . A . Mickelsen, B . E . Murray, D . H . Persing, and B . Swaminathan. 1995 . Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing . J . Clin . Microbiol . 33:2233-2239.
16. Vahaboglu, H., R . Ozturk, H . Akbal, F . Coskunkan, A . Yaman, A . Kaygusuz, H . Lecblebicioglu, I . Balik, K . Aydin, and M . Oktun. 1997 . Widespread detection of PER-1-type extended-spectrum ß-lactamase among nosocomial Acinetobacter and Pseudomonas aeruginosa isolates in Turkey: a nationwide multicenter study . Antimicrob . Agents Chemother . 41:2265-2269.
17. Vatopoulos, A . C., A . Philippon, L . S . Tzouvelekis, Z . Komninou, and N . J . Legakis. 1990 . Prevalence of a transferable SHV-5 type ß-lactamase in clinical isolates of Klebsiella pneumoniae and Escherichia coli in Greece . J . Antimicrob . Chemother . 26:635-648.
18. Weldhagen, G . F., L . Poirel, and P . Nordmann. 2003 . Ambler class A extended-spectrum ß-lactamases in Pseudomonas aeruginosa: novel developments and clinical impact . Antimicrob . Agents Chemother . 47:2385-2392.

Free Online Full-text Article

What Is Genetic Engineering?, What Is Pcr?, What Is Functional Genomics?, What Is MIC?, What Is Salmonella?, o, Microorganism, a, Microbe, n, Microbes, c, Microbiology, e, Bacteriology, a, Staphylococcus, a, Beta lactamase, i, Bacillus, c, Yeasts, n, Bacteroides, s, Bacteriophages, r, Bacteriophages, i, Campylobacter, r, Bactericidal, c, Penicillin, i, S. cerevisiae, i, Fermentations, s, Escherichia coli, n, Microorganism, i, Staphylococcus aureus, c, Bactericidal, i, Campylobacter, o, Microorganism, c, Escherichia coli, e, Escherichia coli, a, Lactobacillus




 

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

Last modified: May 25, 2005