Extended-spectrum β-lactamase TEM-4 in Pseudomonas aeruginosa
1999; Elsevier BV; Volume: 5; Issue: 10 Linguagem: Inglês
10.1111/j.1469-0691.1999.tb00425.x
ISSN1469-0691
AutoresLaurent Poirel, E Ronco, Thierry Naas, Patrice Nordmann,
Tópico(s)Vibrio bacteria research studies
ResumoExtended-spectrum β-lactamases (ESBLs) constitute a growing class of β-lactamases which are often plasmid-mediated and are most commonly expressed in enterobacterial species [1Bush K Jacoby GA Medeiros AA A functional classification scheme for β-lactamases and its correlation with molecular structure.Antimicrob Agents Chemother. 1995; 39: 1211-1233Crossref PubMed Scopus (2039) Google Scholar, 2Jacoby GA Medeiros AA More extended-spectrum β-lactamases. Antimicrob.Agents Chemother. 1991; 35: 1697-1704Crossref PubMed Scopus (635) Google Scholar]. The majority of ESBLs are point mutant derivatives of the narrow-spectrum β-lactamases TEM-1, TEM-2 or SHV-1. They are Ambler class A β-lactamases, hydrolyzing to different extents oxyiminocephalosporins, such as ceftriaxone, cefotaxime and ceftazidime, and monobactams such as aztreonam. The activity of these penicillinases remains inhibited by clavulanic acid [1Bush K Jacoby GA Medeiros AA A functional classification scheme for β-lactamases and its correlation with molecular structure.Antimicrob Agents Chemother. 1995; 39: 1211-1233Crossref PubMed Scopus (2039) Google Scholar]. Such enzymes have rarely been reported in Pseudomonas aeruginosa, only TEM-42 and SHV2a being known in rare isolates in France [3Mugnier P Dubrous S Arlet G Collatz E A TEM-derived extended spectrum β-lactamase in Pseudomonas aeruginosa.Antimicrob Agents Chemother. 1996; 40: 2488-2493PubMed Google Scholar, 4Naas T Philippon L Poirel L Ronco E Nordmann P An SHV-derived extended-spectrum β-lactamase in Pseudomonas aeruginosa.Antimicrob Agents Chemother. 1999; 43: 1281-1284PubMed Google Scholar], and SHV-5 in an isolate from Thailand [5Hawkey P Chanawong A Lulitanond A et al.SHV-5 extended-spectrum β-lactamase from Pseudomonas aeruginosa in Thailand [abstract C174a].in: Program and abstracts of the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA. American Society for Microbiology, Washington DC1998Google Scholar]. PER-1, a class A ESBL not related to TEM or SHV derivatives, however, is widespread in P. aeruginosa isolates from Turkey [6Nordmann P Naas X Sequence analysis of PER-1 extended-spectrum β-lactamase from Pseudomonas aeruginosa and comparison with class A β-lactamases.Antimicrob Agents Chemother. 1994; 38: 104-114Crossref PubMed Scopus (116) Google Scholar, 7Vahaboglu H Ozturk R Aygun G et al.Widespread detection of PER-type extended-spectrum β-lactamase among nosocomial Acinetobacter and Pseudomonas aeruginosa isolates in Turkey: a nationwide multicenter study.Antimicrob Agents Chemother. 1997; 41: 2265-2269Crossref PubMed Google Scholar], while OXA-18, the only class D enzyme conferring an ESBL phenotype, was recently identified in a P. aeruginosa strain from an Italian patient in Paris [8Philippon LN Naas T Bouthors AT Barakett V Nordmann P OXA-18, a class D clavulanic-acid inhibited extended-spectrum β-lactamase from Pseudomonas aeruginosa.Antimicrob Agents Chemother. 1997; 41: 2188-2195PubMed Google Scholar]. Other class D ESBLs whose activity is not clavulanic acid-inhibited, such as the OXA-10 derivatives, OXA-11, OXA-14, OXA-16, and OXA-19, and the OXA-2 derivative, OXA-15, have been reported mostly as plasmid-mediated in P aeruginosa [9Hall LM Livermore D Gur D Akova M Akalin HE OXA-11, an extended-spectrum variant of OXA-10 (PSE-2) β-lactamase from Pseudomonas aeruginosa.Antimicrob Agents Chemother. 1993; 37: 1637-1644Crossref PubMed Scopus (108) Google Scholar, 10Danel F Hall LM Gur D Livermore D OXA-14, another extended-spectrum variant of OXA-10 (PSE-2) β-lactamase from Pseudomonas aeruginosa.Antimicrob Agents Chemother. 1995; 39: 1881-1884Crossref PubMed Scopus (94) Google Scholar, 11Danel F Hall LMC Gur D Livermore DM OXA-15, an extended-spectrum variant of OXA-2 β-lactamase isolated from a Pseudomonas aeruginosa strain.Antimicrob Agents Chemother. 1997; 41: 785-790PubMed Google Scholar, 12Danel F Hall LM Gur D Livemore DM OXA-16; a further extended spectrum variant of OXA-10 β-lactamase from two Pseudomonas aeruginosa isolates.Antimicrob Agents Chemother. 1998; 42: 3117-3122PubMed Google Scholar, 13Mugnier P Casin I Bouthors AT Collatz E Novel OXA-10 derived extended-spectrum β-lactamases selected in vivo or in vitro.Antimicrob Agents Chemother. 1998; 42: 3113-3116PubMed Google Scholar]. In order to detect ESBL-producing P. aeruginosa strains, we have systematically screened all P. aeruginosa strains isolated in our hospitals for ESBL by placing a clavulanic acid-containing disk next to a ceftazidime disk in a double-disk synergy test as suggested for Enterobacteriaceae [14Jarlier V Nicolas MH Fournier G Philippon A Extended broad-spectrum beta-lactamases conferring transferable resistance to newer beta-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns.Rev Infect Dis. 1988; 10: 867-878Crossref PubMed Scopus (1285) Google Scholar]. In December 1996, an image of synergy was detected for P. aeruginosa STEL. This strain was isolated from a urinary tract infection of a man who was transferred to our institution (Raymond Poincaré Hospital) from another suburban hospital of Paris. A strain with a similar resistance phenotype was isolated from a rectal swab of this patient but no other carriers were identified in the same hospital unit in December 1996. P. aeruginosa STEL was identified using the API 32 GN system (bioMérieux, Marcy-l’Etoile, France). Minimum inhibitory concentrations were determined by an agar dilution method as previously described [8Philippon LN Naas T Bouthors AT Barakett V Nordmann P OXA-18, a class D clavulanic-acid inhibited extended-spectrum β-lactamase from Pseudomonas aeruginosa.Antimicrob Agents Chemother. 1997; 41: 2188-2195PubMed Google Scholar] in the presence or absence of a β-lactam inhibitor (clavulanic acid, tazobactam). P. aeruginosa strain STEL was resistant to ticarcillin and piperacillin, but susceptible to ticarcillin/clavulanic acid or piperacillin/tazobactam (Table 1). Resistance towards oxyiminocephalosporins was of low level, especially to ceftazidime. The ceftazidime activity was antagonized by clavulanic acid or tazobactam addition, suggesting the presence of an ESBL.Table 1MICs of β‐lactams for P. aeruginosa isolate STELMICs (mg/L)β‐LactamAntibiotic aloneAntibiotic+ Clavulanic acid (2 mg/L)Antibiotic+ Tazobactam (4 mg/L)Amoxycillin>512>512>512Ticarcillin>5123232Piperacillin3248Cephalothin>512NDNDCefoxitin>512NDNDCeftazidime841Cefotaxime1283216Cefepime822Imipenem4NDNDMeropenem1NDNDMoxalactam16NDNDAztreonam1644ND, not determined. Open table in a new tab ND, not determined. In order to identify this ESBL, dot-blot hybridizations were performed using internal probes for blaPER–1, blaSHV-3, blaTEM and blaOXA-18 [8Philippon LN Naas T Bouthors AT Barakett V Nordmann P OXA-18, a class D clavulanic-acid inhibited extended-spectrum β-lactamase from Pseudomonas aeruginosa.Antimicrob Agents Chemother. 1997; 41: 2188-2195PubMed Google Scholar]. Only the blaTEM probe gave a positive hybridization signal with extracted P. aeruginosa STEL genomic DNA. Using blaTEM specific primers (TEM-1A, 5‘-ATGAGTATTCAA CATTTTCGTGTCG-3’, TEM-1B, 5‘-ACCAATGCTTAATC AGTGAGGCA-3’), an 861-bp PCR product was amplified and sequenced with an Applied Biosystem sequencer (model ABI 377). DNA sequence analysis and deduction of the protein sequence revealed TEM-4. This enzyme, first identified in Escherichia coli [15Sougakoff W Petit A Goussard S Sirot D Bure A Courvalin P Characterization of the plasmid genes blaT-4 and blaT-5 which encode the broad-spectrum beta-lactamases TEM-4 and TEM-5 in Enterobacteriaceae.Gene. 1989; 78: 339-348Crossref PubMed Scopus (63) Google Scholar, 16Paul GC Gerbaud G Bure A Philippon AM Pangon B Courvalin P TEM-4, a new plasmid-mediated β-lactamase that hydrolyzes broad-spectrum cephalosporins in a clinical isolate of Escherichia coli.Antimicrob Agents Chemother. 1989; 33: 1958-1963Crossref PubMed Scopus (35) Google Scholar], differs from the narrow-spectrum β-lactamase TEM-1 by four amino acid substitutions, L21F, E104K, G238S, and T265M. The E104K and G238S changes were shown to be responsible for the extended hydrolysis spectrum of TEM-4 that was initially considered to indicate a cefotaximase rather than a ceftazidimase [15Sougakoff W Petit A Goussard S Sirot D Bure A Courvalin P Characterization of the plasmid genes blaT-4 and blaT-5 which encode the broad-spectrum beta-lactamases TEM-4 and TEM-5 in Enterobacteriaceae.Gene. 1989; 78: 339-348Crossref PubMed Scopus (63) Google Scholar, 16Paul GC Gerbaud G Bure A Philippon AM Pangon B Courvalin P TEM-4, a new plasmid-mediated β-lactamase that hydrolyzes broad-spectrum cephalosporins in a clinical isolate of Escherichia coli.Antimicrob Agents Chemother. 1989; 33: 1958-1963Crossref PubMed Scopus (35) Google Scholar]. Since blaTEM–4 was found to be plasmid-located in E. coli, four different plasmid DNA extraction methods were used for P. aeruginosa STEL [8Philippon LN Naas T Bouthors AT Barakett V Nordmann P OXA-18, a class D clavulanic-acid inhibited extended-spectrum β-lactamase from Pseudomonas aeruginosa.Antimicrob Agents Chemother. 1997; 41: 2188-2195PubMed Google Scholar]. None of these methods yielded plasmid DNA from this P. aeruginosa strain. Mating-out experiments using either P. aeruginosa PU21 or an in vitro obtained ciprofloxacin-resistant E. coli JM109 mutant as recipient strains also failed [8Philippon LN Naas T Bouthors AT Barakett V Nordmann P OXA-18, a class D clavulanic-acid inhibited extended-spectrum β-lactamase from Pseudomonas aeruginosa.Antimicrob Agents Chemother. 1997; 41: 2188-2195PubMed Google Scholar]. The blaTEM–4 gene is therefore likely to be chromosome-borne in P. aeruginosa STEL, just as it is the case of other clavulanic acid-inhibited ESBLs in P aeruginosa such as blaSHV-2a, blaSHV-5, blaOXA-18, and blaPER-1 (in the initial strain PERO) [4Naas T Philippon L Poirel L Ronco E Nordmann P An SHV-derived extended-spectrum β-lactamase in Pseudomonas aeruginosa.Antimicrob Agents Chemother. 1999; 43: 1281-1284PubMed Google Scholar, 5Hawkey P Chanawong A Lulitanond A et al.SHV-5 extended-spectrum β-lactamase from Pseudomonas aeruginosa in Thailand [abstract C174a].in: Program and abstracts of the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA. American Society for Microbiology, Washington DC1998Google Scholar, 6Nordmann P Naas X Sequence analysis of PER-1 extended-spectrum β-lactamase from Pseudomonas aeruginosa and comparison with class A β-lactamases.Antimicrob Agents Chemother. 1994; 38: 104-114Crossref PubMed Scopus (116) Google Scholar, 8Philippon LN Naas T Bouthors AT Barakett V Nordmann P OXA-18, a class D clavulanic-acid inhibited extended-spectrum β-lactamase from Pseudomonas aeruginosa.Antimicrob Agents Chemother. 1997; 41: 2188-2195PubMed Google Scholar]. A lower level of resistance to oxyiminocephalosporins was found in P. aeruginosa STEL as compared to that observed in E. coli [16Paul GC Gerbaud G Bure A Philippon AM Pangon B Courvalin P TEM-4, a new plasmid-mediated β-lactamase that hydrolyzes broad-spectrum cephalosporins in a clinical isolate of Escherichia coli.Antimicrob Agents Chemother. 1989; 33: 1958-1963Crossref PubMed Scopus (35) Google Scholar]. This may result from either the absence of plasmid location of blaTEM-4 in P. aeruginosa STEL, or a weaker expression of this gene in P. aeruginosa, or both. With the use of 5'CS (5‘-GGCATCCAAGCAGCAAG-3’) and 3'CS (5‘-AAGCAGACTTGACC TGA-3’) specific primers for class 1 integrons [17Naas T Sougakoff W Casetta A Nordmann P OXA-20, a novel class D β-lactamase, and its integron from Pseudomonas aeruginosa.Antimicrob Agents Chemother. 1998; 42: 2074-2083PubMed Google Scholar], a 1400-bp PCR-amplified fragment was obtained from P. aeruginosa STEL genomic DNA which did not hybridize with the internal probe for blaTEM. Thus, the location of blaTEM–4 in a typical class 1 integron was rejected as the result of blaTEM–4 acquisition in P aeruginosa STEL. This study constitutes the second report of a TEM-derivative ESBL in P. aeruginosa. Together with previous reports, it indicates that P aeruginosa strains may become a hidden reservoir for ESBL genes, as is already known to be the case for oxacillinase extended-spectrum derivatives [18Nordmann P Guibert M Extended-spectrum β-lactamases in Pseudomonas aeruginosa.J Antimicrob Chemother. 1998; 42: 128-131Crossref PubMed Google Scholar]. In the case of blaTEM–4, its presence in P. aeruginosa may result either from point mutations of a pre-existing blaTEM–1 gene or from transfer of blaTEM–4, perhaps from an enterobacterial species. From a clinical point of view, it is worthwhile mentioning that ESBLs are difficult to detect in P aeruginosa. Their detection by the double-disk synergy test may be rendered more efficient by placing ceftazidime and clavulanic acid disks closer to each other. ‘Candidate strains’ for clavulanic acid-inhibited ESBL-producing P. aeruginosa are ticarcillin and ceftazidime resistant, but susceptible to ticarcillin–clavulanate.
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