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. 2002 Nov;46(11):3624–3626. doi: 10.1128/AAC.46.11.3624-3626.2002

Clinical Strain of Pseudomonas aeruginosa Carrying a blaTEM-21 Gene Located on a Chromosomal Interrupted TnA Type Transposon

Véronique Dubois 1,*, Corinne Arpin 1, Patrick Noury 2, Claudine Quentin 1
PMCID: PMC128703  PMID: 12384376

Abstract

A clinical isolate of Pseudomonas aeruginosa was found to produce a clavulanic acid-inhibited extended-spectrum β-lactamase with a pI of 6.4. PCR, cloning, and sequencing experiments showed that the corresponding blaTEM-21 gene was part of a chromosomally located Tn801 transposon disrupted by an IS6100 element and adjacent to an aac(3)-II gene.


Most extended-spectrum β-lactamases (ESBLs) produced by enterobacteria derive from the common plasmid-mediated penicillinases TEM-1, TEM-2, and SHV-1 (14). ESBLs have recently emerged in Pseudomonas aeruginosa, where they still remain infrequent. In this bacterial species, ESBLs are mainly OXA derivatives belonging to Ambler class D or newly emerging class A β-lactamases such as PER-1 (23) and VEB-1 (9). However, occasionally some enzymes of the TEM or SHV families, previously (TEM-4, TEM-24, and SHV-2a) or newly (TEM-42) described, have been also found (11-13, 19). These observations suggest that ESBLs widespread in the Enterobacteriaceae family may be increasingly found in P. aeruginosa (13), which could also be a reservoir for the dissemination of this kind of enzyme. We report here the production of an unusual TEM derivative, TEM-21, in a clinical strain of P. aeruginosa.

(This work was presented in part at the 40th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 17 to 20 September 2000.)

P. aeruginosa Pa141 was isolated in 1997 in a private laboratory in Bordeaux, France, from the urinary tract of a 68-year-old woman living in a nursing home, with Alzheimer's disease and carrying an indwelling urinary tract catheter. This strain belonged to serogroup P2, and by the disk diffusion method it was resistant to all antipseudomonal β-lactams except for imipenem. A marked synergistic effect between clavulanic acid and cefotaxime, ceftazidime, or aztreonam was observed, suggesting the presence of an ESBL. Indeed, the isolate remained susceptible to the combinations of ticarcillin and clavulanic acid and of piperacillin and tazobactam. In addition, Pa141 was resistant to gentamicin, tobramycin, netilmicin, ciprofloxacin, and fosfomycin. These data were confirmed by MIC determination (Table 1) by an agar dilution method in Mueller-Hinton medium according to official guidelines (http://www.sfm.asso.fr). Isoelectric focusing of crude β-lactamase extracts was performed on a pH 3.5 to 10 Ampholine polyacrylamide gel and revealed by the iodine procedure, with benzylpenicillin (75 μg/ml) as substrate. By this method the Pa141 strain produced a single β-lactamase of pI 6.4 cofocusing with the TEM-21 reference enzyme (22).

TABLE 1.

Antimicrobial susceptibilities of the clinical strain Pa141, E. coli XL1Blue harboring the recombinant plasmid pMF6, and the reference strain

Antimicrobial agent(s)a MIC (μg/ml) for:
P. aeruginosa Pa141 E. coli XL1Blue (pMF6) E. coli XL1Blue
Ticarcillin >512 >512 4
Ticarcillin + CLA 32 128 4
Ceftazidime 32 64 0.2
Ceftazidime + CLA 4 0.2 0.2
Cefotaxime 256 64 <0.1
Cefotaxime + CLA 256 <0.1 <0.1
Cefsulodin 512 >512 32
Cefepime 64 8 <0.1
Cefpirome 128 256 <0.1
Piperacillin 128 256 2
Piperacillin + TZB 8 1 1
Imipenem 2 0.2 0.2
Aztreonam 32 32 <0.1
Aztreonam + CLA 8 <0.1 <0.1
Gentamicin 512 >512 0.5
Tobramycin 128 32 0.5
Netilmicin >512 32 0.5
Amikacin 16 2 2
a

CLA, clavulanic acid, 2 μg/ml; TZB, tazobactam, 4 μg/ml.

Total DNA of P. aeruginosa Pa141 was extracted as previously described (18) and subjected to PCR amplification with primers specific for the blaTEM genes (22), and the PCR product was sequenced on both strands with the D Rhodamine dye terminator kit (Perkin-Elmer, Courtaboeuf, France) and an automatic sequencer (ABI 377; Perkin-Elmer). The nucleotide sequence of the ESBL-encoding gene differed from that of the blaTEM-21 gene previously described (22) by two silent mutations at positions 369 (C→T) and 624 (G→A) according to Sutcliffe's numbering system (21). The TEM-21 protein derives from the parental TEM-2 enzyme, like TEM-24 (12) and TEM-42 (11), two other ESBLs found in P. aeruginosa. TEM-21 differs from TEM-2 by three amino acid substitutions, Glu104→Lys, His153→Arg, and Gly238→Ser, according to the numbering system of Ambler et al. (2). First described for a Klebsiella pneumoniae isolate in Tunisia (3), TEM-21 is rarely encountered and is much less frequent than TEM-24 and TEM-3 in France (7). However, the blaTEM-21 gene has been already detected and sequenced in a Morganella morganii strain (22) from the same region where Pa141 was recovered.

Transfer resistance by conjugation to an azide-resistant strain of Escherichia coli HB101 or a rifampin-resistant mutant of P. aeruginosa ATCC 27853, by the most efficient filter mating technique (6), did not yield any transconjugants (<10−8). Despite repeated attempts with an alkaline-lysis method (4) and the Qiagen (Courtaboeuf, France) plasmid DNA Midi kit, plasmid DNA analysis of P. aeruginosa Pa141 did not show any plasmid, and transformation by electroporation of plasmid DNA extract into E. coli HB101 was unsuccessful. A Southern blot hybridization with total DNA of Pa141 and a blaTEM probe argued for the chromosomal location of the blaTEM-21 gene in this strain (data not shown). Then, the whole-cell DNA of Pa141 was totally restricted by HindIII and ligated into the HindIII site of pBK-CMV cloning vector. E. coli XL1Blue strains harboring the recombinant plasmids were selected on Mueller-Hinton agar plates containing 100 μg of ampicillin/ml and 50 μg of kanamycin/ml and exhibited the β-lactam and aminoglycoside resistance pattern of Pa141, by MIC determination (Table 1) and isoelectric focusing. After plasmid DNA extraction, a double-restriction digestion with HindIII and PstI enzymes followed by electrophoresis on an 0.8% agarose gel allowed us to evaluate the size of the large fragment (ca 6.7 kb) inserted in the recombinant plasmid pMF6.

Genes encoding the TEM type ESBLs are supposed to be located on transposons of the TnA family as are those for the TEM-1 and TEM-2 parental enzymes. However, while the plasmid or chromosomal origin of these genes is usually determined, their precise genetic location is rarely specified (10). In order to analyze the genetic environment of the blaTEM-21 gene in Pa141, a part of the recombinant plasmid pMF6 was sequenced with laboratory-designed primers. The sequences immediately upstream and downstream from the blaTEM-21 gene exhibited 100% DNA identity with a part of transposon Tn801 from P. aeruginosa (5) (Fig. 1). Indeed, the right side included the IRR (inverted repeat) of the transposon and the left one contained part of the resolvase gene (tnpR, 305 pb). Tn801 is closely related to Tn3 and comprises the blaTEM-2 gene. The resolvase gene of Tn801 was truncated or simply interrupted by the insertion sequence (IS) IS6100 (858 bp sequenced of 880 bp, up to the HindIII site present in IS6100) (Fig. 1). When TEM type ESBL-encoding genes have been situated on transposons, the TnA elements have been generally no longer mobile, due to their disruption and subsequent loss of transposition functions (10). Likewise, IS6100 has been already found in plasmids, transposons, and integrons, where it frequently truncates genes (15-17, 20). At the 3′ end of Tn801 the expected IRR was found adjacent to an aac(3)-II (also called aacC2) gene which encodes a 3-N-aminoglycoside acetyltransferase conferring gentamicin, tobramycin, and netilmicin resistance. The gene and the 34 nucleotides between the gene and the recombination point were identical to those previously described for the Enterobacteriaceae (1, 24) (Fig. 1). The −35 sequence of some aac(3)-II genes was found to be complemented by an IS140 element inserted 43 bp upstream from the gene, creating an improved promoter structure (1). With the software available at the Baylor College of Medicine Search Launcher website (http://searchlauncher.bcm.tmc.edu), the analysis of the promoter sequences of the present aac(3)-II gene indicated that several sequences within the 5′ end of Tn801 may correspond to putative promoters, including one highly homologous (94%) to E. coli promoter sequences for σ70 factor (regions −35[TTGATT] and −10[TTTAAA]) (Fig. 1). ISs, but much less commonly transposons, are known to provide mobile promoters for prokaryotic gene expression. Another open reading frame of 321 nucleotides was found 12 bp downstream from the 3′ end of the aac(3)-II gene. Except for a Tyr→His substitution at position 84, this open reading frame encoded a protein of 106 amino acids identical to that described in part for plasmids pWP14A and pWP113A, also separated by 12 bp from the same aac(3)-II gene (1). A 905-bp sequence at the 3′ end of this gene was determined but did not show any significant homology (<39%) with sequences compiled in the GenBank database. The blaTEM-21 and aac(3)-II genes are mostly encountered in the Enterobacteriaceae, and they seemed chromosomally located in Pa141, thus supporting the hypothesis that a plasmid derived from enterobacteria became integrated into Pa141 chromosomal DNA either by homologous recombination or by IS- or transposon-mediated specific cointegration (8).

FIG. 1.

FIG. 1.

Schematic representation of the 6.7-kb insert of the recombinant plasmid pMF6. The solid lines represent the sequenced region with the various genes boxed. The horizontal arrows indicate the translation orientation. The open arrowhead represents the terminal inverted repeat of IS6100, and the striped arrowhead represents the inverted repeat of Tn801. Details on the nucleotide sequence of the aac(3)-II promoter region and the recombination point are shown below. The double arrow indicates the size of the IS and transposon homologous regions. The dotted lines indicate the unsequenced fragment, and dashes are used to indicate that the corresponding sequences are not to scale. RBS, ribosomal binding site.

This study is the first description of the blaTEM-21 genetic environment and the presence of this gene in a clinical isolate of P. aeruginosa, highlighting once more the broad exchange of resistance genes from Enterobacteriaceae to P. aeruginosa strains.

Nucleotide sequence accession number.

The nucleotide sequences reported in this work are available in the GenBank nucleotide database under the accession no. AF466526.

Acknowledgments

We thank Catherine André and Cécile Frigo for technical assistance.

This work was supported by grants from the French Network on β-lactamase study and from the Ministère de l'Education Nationale et de la Recherche (EA-525), Université de Bordeaux 2, Bordeaux, France.

REFERENCES

  • 1.Allmansberger, R., B. Brau, and W. Piepersberg. 1985. Genes for gentamicin-(3)-N-acetyl-transferases III and IV. II. Nucleotide sequences of three AAC(3)-III genes and evolutionary aspects. Mol. Gen. Genet. 198:514-520. [DOI] [PubMed] [Google Scholar]
  • 2.Ambler, R. P., A. F. Coulson, J. M. Frere, J. M. Ghuysen, B. Joris, M. Forsman, R. C. Levesque, G. Tiraby, and S. G. Waley. 1991. A standard numbering scheme for the class A β-lactamases. Biochem. J. 276:269-270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Ben Redjeb, S., G. Fournier, C. Mabilat, A. Ben Hassen, and A. Philippon. 1990. Two novel transferable extended-spectrum β-lactamases from Klebsiella pneumoniae in Tunisia. FEMS Microbiol. Lett. 55:33-38. [DOI] [PubMed] [Google Scholar]
  • 4.Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Burland, V., Y. Shao, N. T. Perna, G. Plunkett, H. J. Sofia, and F. R. Blattner. 1998. The complete DNA sequence and analysis of the large virulence plasmid of Escherichia coli O157:H7. Nucleic Acids Res. 26:4196-4204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Courvalin, P., F. Goldstein, A. Philippon, and J. Sirot. 1985. L'antibiogramme. MPC-VIDEOM, Paris, France.
  • 7.De Champs, C., D. Sirot, C. Chanal, R. Bonnet, J. Sirot, and The French Study Group. 2000. A 1998 survey of extended-spectrum β-lactamases in Enterobacteriaceae in France. Antimicrob. Agents Chemother. 44:3177-3179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Galas, D. J., and M. Chandler. 1989. Bacterial insertion sequences, p. 109-162. In D. E. Berg and M. M. Howe (ed.), Mobile DNA. American Society for Microbiology, Washington, D.C.
  • 9.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 β-lactamase in Pseudomonas aeruginosa in Thailand. Clin. Infect. Dis. 34:603-611. [DOI] [PubMed] [Google Scholar]
  • 10.Heritage, J., P. M. Hawkey, N. Todd, and I. J. Lewis. 1992. Transposition of the gene encoding a TEM-12 extended-spectrum β-lactamase. Antimicrob. Agents Chemother. 36:1981-1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Marchandin, H., H. Jean-Pierre, C. De Champs, D. Sirot, H. Darbas, P. F. Perigault, and C. Carriere. 2000. Production of a TEM-24 plasmid-mediated extended-spectrum β-lactamase by a clinical isolate of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 44:213-216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Mugnier, P., P. Dubrous, I. Casin, G. Arlet, and E. Collatz. 1996. A TEM-derived extended-spectrum β-lactamase in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 40:2488-2493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Naas, T., L. Philippon, L. Poirel, E. Ronco, and P. Nordmann. 1999. An SHV-derived extended-spectrum β-lactamase in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 43:1281-1284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Nordmann, P. 1998. Trends in β-lactam resistance among Enterobacteriaceae. Clin. Infect. Dis. 27:S100-S106. [DOI] [PubMed] [Google Scholar]
  • 15.Partridge, S. R., H. J. Brown, H. W. Stokes, and R. M. Hall. 2001. Transposons Tn1696 and Tn21 and their integrons In4 and In2 have independent origins. Antimicrob. Agents Chemother. 45:1263-1270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Partridge, S. R., G. D. Recchia, H. W. Stokes, and R. M. Hall. 2001. Family of class 1 integrons related to In4 from Tn1696. Antimicrob. Agents Chemother. 45:3014-3020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Ploy, M. C., F. Denis, P. Courvalin, and T. Lambert. 2000. Molecular characterization of integrons in Acinetobacter baumannii: description of a hybrid class 2 integron. Antimicrob. Agents Chemother. 44:2684-2688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Poirel, L., T. Naas, D. Nicolas, L. Collet, S. Bellais, J. D. Cavallo, and P. Nordmann. 2000. Characterization of VIM-2, a carbapenem-hydrolyzing metallo-β-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Antimicrob. Agents Chemother. 44:891-897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Poirel, L., E. Ronco, T. Naas, and P. Nordmann. 1999. Extended spectrum β-lactamase TEM-4 in Pseudomonas aeruginosa. Clin. Microbiol. Infect. 5:651-652. [DOI] [PubMed] [Google Scholar]
  • 20.Preston, K. E., C. C. Radomski, and R. A. Venezia. 1999. The cassettes and 3′ conserved segment of an integron from Klebsiella oxytoca plasmid pACM1. Plasmid 42:104-114. [DOI] [PubMed] [Google Scholar]
  • 21.Sutcliffe, J. G. 1978. Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proc. Natl. Acad. Sci. USA 75:3737-3741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Tessier, F., C. Arpin, A. Allery, and C. Quentin. 1998. Molecular characterization of a TEM-21 β-lactamase in a clinical isolate of Morganella morganii. Antimicrob. Agents Chemother. 42:2125-2127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Vahaboglu, H., R. Ozturk, G. Aygun, F. Coskunkan, A. Yaman, A. Kaygusuz, H. Leblebicioglu, I. Balik, K. Aydin, and M. Otkun. 1997. Widespread detection of PER-1-type extended-spectrum β-lactamases among nosocomial Acinetobacter and Pseudomonas aeruginosa isolates in Turkey: a nationwide multicenter study. Antimicrob. Agents Chemother. 41:2265-2269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Vliegenthart, J. S., P. A. Ketelaar-van Gaalen, and J. A. van de Klundert. 1989. Nucleotide sequence of the aacC2 gene, a gentamicin resistance determinant involved in a hospital epidemic of multiply resistant members of the family Enterobacteriaceae. Antimicrob. Agents Chemother. 33:1153-1159. [DOI] [PMC free article] [PubMed] [Google Scholar]

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