Emergence of imipenem resistance in Klebsiella pneumoniae owing to combination of plasmid-mediated CMY-4 and permeability alteration

Van Thi Bao Caoa, Guillaume Arletb, Britt-Marie Ericssonc, Ann Tammelinc, Patrice Courvalina and Thierry Lamberta,d,*

a Unité des Agents Antibactériens, Institut Pasteur, 75724 Paris Cedex 15, France; b Service de Bactériologie, Hôpital Tenon, 75970 Paris Cedex 20, France; c University Hospital, Uppsala, Sweden; d Centre d'Etude Pharmaceutiques, Châtenay–Malabry, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Klebsiella pneumoniae BM2974 isolated from an abdominal abcess was resistant to high concentrations of all available ß-lactams, including recently developed third-generation cephalosporins and carbapenems. Isoelectric focusing of ß-lactamases and amplification, cloning and sequencing of the corresponding genes, together with conjugation and transformation experiments, indicated that, in addition to the chromosomally encoded ß-lactamase, the strain produced three plasmid-mediated ß-lactamases with pIs of 5.4, 8.2 and 9.0, which corresponded to TEM-1, SHV-5 and AmpC-type CMY-4, respectively. Strain BM2974 also lacked a major outer membrane protein of c. 40 kDa which was present in the spontaneous imipenem-susceptible revertant BM2974-1. We suggest that imipenem resistance in strain BM2974 is attributable to production of CMY-4 ß-lactamase combined with permeability alteration.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Carbapenems are the ß-lactams with the broadest spectrum of antibacterial activity. Resistance to these antibiotics has emerged in various Gram-negative bacterial genera via two major mechanisms: (i) production of carbapenem-hydrolysing ß-lactamases (class A serine ß-lactamases or class B metallo-enzymes)1 or (ii) high-level production of chromosomal AmpC cephalosporinases combined with decreased outer membrane permeability in Enterobacter aerogenes,24 Enterobacter cloacae,5,6 Providencia rettgerii,7 Proteus mirabilis8 and Citrobacter freundii.9

Recently, synthesis of plasmid-mediated AmpC-type ß-lactamase ACT-1 combined with lack of a major porin was found to be responsible for imipenem resistance in clinical isolates of Klebsiella pneumoniae.10 Similarly, production of SHV2 ß-lactamase was found to confer low-level resistance to imipenem in a K. pneumoniae strain deficient in a major 40 kDa outer membrane protein.11 In this report, we studied the mechanisms responsible for resistance to all available ß-lactams, including imipenem, in a clinical isolate of K. pneumoniae resistant to multiple classes of drugs.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains, plasmids and growth conditions

K. pneumoniae BM2974 was isolated in 1998 in Sweden from the peritoneal abscess of a 61-year-old female patient. She had been hospitalized previously for peritonitis in Bombay, where she had been treated with ceftazidime, ciprofloxacin and vancomycin. Six weeks after arrival in Sweden, the patient developed a colonic perforation with abscess. The abscess was drained and the patient recovered.

The origins and properties of the other strains used in this study are shown in Table IGo. All the strains were grown in brain heart infusion (BHI) broth and agar (Difco, Detroit, MI, USA) at 37°C. The MICs of ß-lactams were determined by a standard agar dilution method12 with 104 cfu/spot on Mueller–Hinton agar (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France). Antibiotic susceptibility was tested by a disc diffusion method on Mueller– Hinton agar.12


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Table I. Strains and plasmids
 
Transfer of resistance

Transfer of resistance determinants from K. pneumoniae BM2974 by conjugation or by transformation with purified plasmid DNA was carried out as described13,14 using Escherichia coli C600-Rif and E. coli JM83 as recipients, respectively. Transconjugants resistant to ß-lactams were selected on BHI agar supplemented with rifampicin 250 mg/L plus either ampicillin 100 mg/L or ceftazidime 2 mg/L, while those resistant to tobramycin were selected on BHI agar supplemented with rifampicin 250 mg/L plus tobramycin 5 mg/L. Transformants encoding an AmpC ß-lactamase were selected on BHI agar supplemented with cefoxitin 32 mg/L, which is a poor substrate for penicillinases.

Isoelectric focusing of ß-lactamases

Bacteria grown in BHI broth were harvested in the exponential phase of growth and cell-free extracts were prepared by sonication.15 Analytical isoelectric focusing (IEF) was performed on ampholine polyacrylamide gels (pH 3.5–10) for 18 h at 400 V, using a Multiphor apparatus (Pharmacia Biotech, Saclay, France). ß-Lactamase activity was detected directly on the gel by the nitrocefin procedure.16 The isoelectric point (pI) of each enzyme was determined by comparison with reference ß-lactamases: TEM-1 (pBR322, pI 5.4), TEM-3 (pCFF04, pI 6.3), SHV-2 (pUD20, pI 7.7), SHV-5 (pASS-2, pI 8.2) and CMY-2b (pSENF, pI 9.0).

DNA amplification

The primers specific for TEM, SHV and AmpC genes were as follows: TEM-L (5'-ATGAGTATTCAACATTT-3') and TEM-R (5'-TTACCAATGCTTAATCA-3'); OS5 (5'-TTATCTCCCTGTTAGCCACC-3') and OS6-R (5'-GATTTGCTGATTTCGCTCGG-3'); AmpC A1 (5'-GGAATTCCTWTGCTGCGCBCTGCTGCT-3') and AmpC A2 (5'-CGGGATCCCTGCCAGTTTTGATAAAA-3').17 These primers gave PCR products that comprised bp 1–780 (where bp 1 is the first base in the gene), bp 23–818 and bp 17–458, respectively, which included the regions known to support critical mutations affecting the activity of TEM and SHV enzymes. Amplification was performed in 100 µL reaction mixtures consisting of 1 x Pfu DNA polymerase buffer, 1.5 mM of MgCl2, 200 µM of deoxynucleoside triphosphates, 50 pmol of each primer, 2 IU of Pfu DNA polymerase (Stratagene, La Jolla, CA, USA) and 25 ng of DNA in a GeneAmp PCR system 2400 (Perkin–Elmer Cetus, Norwalk, CT, USA). Total DNA was used as a template for PCR.

Plasmid DNA preparation and cloning

Plasmid DNA from wild strains and transconjugants was obtained as described previously,18 whereas DNA from recombinant plasmids was extracted with a commercial Wizard Plus Miniprep DNA Purification System (Promega, Madison, WI, USA). EcoRI-digested fragments were cloned as described by Sambrook et al.14 into pBGS18 with selection on kanamycin 50 mg/L. Recombinant plasmids were introduced by transformation into Salmonella enterica BM4411 and the transformants were selected on BHI agar containing cefotaxime 4 mg/L and kanamycin 50 mg/L.

Sequence analysis

DNA sequencing was performed by the dideoxynucleotide chain termination method19 with [{alpha}-35S]dATP (Amersham, Cleveland, OH, USA) and the T7 Sequenase version 2.0 DNA sequencing kit (Amersham). Nucleotide and amino acid sequences were analysed and compared with sequences in the GenBank, EMBL and Swiss-Prot databases using the FASTA program (Genetics Computer Group software).

Outer membrane protein analysis

Outer membrane proteins were extracted by solubilization in 0.01 M HEPES containing 2% Triton X-100 of cell envelopes obtained by sonication of bacteria grown in BHI broth, as described previously.20 The proteins were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) in a running buffer of 0.02 M Tris, 0.19 M glycine and 0.1% SDS (pH 8.3) with a 12% acrylamide, 0.12% bis-acrylamide gel and stained with Coomassie Blue.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Antibiotic susceptibility of K. pneumoniae BM2974

K. pneumoniae BM2974 was highly resistant to all commercially available ß-lactams, including imipenem and aztreonam (Table IIGo). The strain was also resistant to amikacin, gentamicin, netilmicin and tobramycin as a result of synthesis of an aminoglycoside 6'-N-acetyltransferase, as evidenced by the differential activity of 2'- and 6'- N-ethylnetilmicin,21 and to streptomycin, tetracycline–minocycline, chloramphenicol, trimethoprim, sulphonamides and ciprofloxacin. An imipenem-susceptible variant, BM2974-1, was obtained by subculture of K. pneumoniae BM2974 for c. 40 generations in the absence of antibiotic.


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Table II. MICs (mg/L) of various ß-lactams against K. pneumoniae and E. coli strains
 
Transfer of resistance to antibiotics

E. coli transconjugants could be classified into three groups on the basis of their plasmid content and antibiotic resistance (Table IIIGo): (i) those that harboured plasmid pIP1859 and were resistant to kanamycin and amino- and acylureido-penicillins, with susceptibility to the latter being restored in the presence of clavulanic acid; (ii) those that harboured plasmid pIP1860 and were resistant to gentamicin and structurally related aminoglycosides, trimethoprim, cephalothin and ceftazidime—in this group there was synergy between clavulanic acid and third-generation cephalosporins, indicating the production of an extended-spectrum ß-lactamase; (iii) those that were resistant in addition to high levels of oxyimino- and 7-methoxy-cephalosporins (cephamycins), streptomycin, sulphonamides, tetracycline, trimethoprim and chloramphenicol but not to imipenem, and containing plasmids pIP1860 and pIP1861. Transformation of E. coli JM83 with plasmid DNA from E. coli C600, from transconjugants containing pIP1860 and pIP1861, with selection on cefoxitin, indicated that pIP1861 was responsible for resistance to oxyimino-cephalosporins and cephamycins, chloramphenicol, streptomycin, sulphonamides, tetracycline and trimethoprim. Combination of clavulanic acid with other ß-lactams did not show synergy. Attempts to transfer pIP1861 by conjugation from these transformants were unsuccessful.


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Table III. Properties of the E. coli derivatives
 
Characterization of ß-lactamases

Analysis by IEF of the ß-lactamases in K. pneumoniae BM2974 and in the imipenem-susceptible derivative BM2974-1 indicated the presence of four enzymes with pIs of 5.4, 7.7, 8.2 and 9.0 (data not shown). The ß-lactamase with a pI of 7.7 was present only in K. pneumoniae BM2974 and BM2974-1 but not in the E. coli derivatives; it probably corresponds to the chromosomally encoded speciesspecific LEN-1 enzyme of K. pneumoniae.22 The ß-lactamases with pI of 5.4, 8.2 and 9.0 were detected in E. coli C600 (pIP1859), E. coli C600 (pIP1860) and E. coli JM83 (pIP1861), respectively, and correspond to TEM-1, SHV-5 and AmpC-type ß-lactamases, respectively.

PCR and sequencing of blaTEM-1 and blaSHV-5

The presence of structural genes for TEM and SHV ß- lactamases in K. pneumoniae BM2974 was confirmed by PCR with specific primers. blaTEM was present in E. coli C600 (pIP1859) and blaSHV in E. coli C600 (pIP1860); sequencing of the PCR products indicated identity with blaTEM-1 and blaSHV-5, respectively. blaCMY was detected by PCR in E. coli JM83 (pIP1861) and sequencing of a 484 bp internal fragment indicated 99% identity with blaCMY2.

Cloning and sequence determination of the ampC gene

An 11 kb EcoRI fragment of pIP1861 was cloned into pBGS18 and introduced by transformation into S. enterica BM4411, which is devoid of ß-lactamase; the sequence of 1913 adjacent bp was determined. Analysis of the sequence revealed an open reading frame of 1146 nucleotides corresponding to a deduced protein of 381 amino acids with 100% identity to CMY-4 ß-lactamase. This enzyme was first found in a P. mirabilis isolate from Tunisia and originates from the chromosome of C. freundii.17 Two silent mutations, A661->C and A1140->G, were present in blaCMY-4 from K. pneumoniae BM2974.

Analysis of outer membrane proteins

The fact that the ß-lactamase content of the imipenemsusceptible derivative BM2974-1 was indistinguishable from that of K. pneumoniae BM2974 suggested that imipenem resistance in the parental strain could result, at least in part, to alteration in permeability. Analysis of the outer membrane proteins of these strains by SDS–PAGE (FigureGo) revealed the lack of a major outer membrane protein of 40 kDa in K. pneumoniae BM2974 relative to BM2974-1. This membrane protein, which was also detected in the wild strain of K. pneumoniae BM4441 used as a reference for protein profile, has a greater contribution to imipenem resistance than to resistance to the other ß-lactams (Table IIGo)23 and could correspond to OmpK36, which is known play a role in permeability to carbapenems,23 and is homologous to OmpC of E. coli.



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Figure. Outer membrane proteins of K. pneumoniae strains. Outer membrane proteins were separated by SDS–PAGE in 12% acrylamide gel. Lane 1, K. pneumoniae BM4441; lane 2, K. pneumoniae BM2974; lane 3, K. pneumoniae BM2974-1; lane 4, molecular size standards. The 40 kDa protein in BM4441 and BM2974-1 is indicated by an arrow.

 
Imipenem is usually active against K. pneumoniae that produce extended-spectrum ß-lactamases, including porin-deficient variants. It is also active against isolates producing plasmid-mediated AmpC-type ß-lactamases.24,25 Increased resistance to cephalosporins conferred by permeability alteration has been demonstrated in several studies. The MICs of cefamadole and cefoxitin were increased for strains expressing a broad-spectrum ß-lactamase and lacking a c. 40 kDa outer membrane protein.26 Cefoxitin was used to select mutants of SHV-1 lacking a c. 40 kDa outer membrane protein.27 Loss of a 41 kDa outer membrane protein in K. pneumoniae expressing TEM-2 resulted in resistance to cefoperazone–sulbactam.28 Resistance to cefotaxime and cefoxitin was observed in K. pneumoniae producing SHV-5 with altered 35–40 kDa outer membrane proteins.29 However, in all these instances, resistance to imipenem remained low.

Imipenem resistance in K. pneumoniae due to loss of a 42 kDa outer membrane protein combined with production of ACT-1, an AmpC ß-lactamase originating in E. cloacae, has been reported recently.10 Thus, loss of an outer membrane protein or acquisition of an AmpC ß-lactamase is not sufficient to confer carbapenem resistance upon K. pneumoniae; the two mechanisms have to be combined in order to achieve detectable resistance. Increased expression of the ampC gene of C. freundii, which encodes CMY ß-lactamase, combined with decreased permeability to carbapenems was responsible for carbapenem resistance in this bacterium, although the authors did not detect significant carbapenemase activity against imipenem or meropenem.9 In contrast, BRL 42715 was found to block carbapenem resistance in K. pneumoniae strains deficient in porins and producing an AmpC-type enzyme.23 These results suggest that a small amount of carbapenem, at the limit of detection, could be hydrolysed.

In K. pneumoniae BM2974, synthesis of three plasmid-mediated ß-lactamases, including AmpC CMY-4, together with alteration in permeability led to resistance to all available ß-lactams. The plasmids also conferred resistance to all commercially available aminoglycosides, trimethoprim, sulphonamides, chloramphenicol, tetracyclines and fluoroquinolones. Fortunately, isolation of the patient prevented dissemination of this highly multiresistant strain.


    Acknowledgments
 
We thank M. Popoff for the gift of S. enterica 4441, and M. Chauvel, G. Gerbaud and S. Magnet for technical advice. This work was supported in part by a Bristol-Myers Squibb Unrestricted Biomedical Research Grant in Infectious Diseases and a grant of UFR St Antoine. V.C. is the recipient of a doctoral fellowship from the Réseau International des Instituts Pasteur et Instituts Associés.


    Notes
 
* Corresponding author. Tel: +33-1-45-68-83-21; Fax: +33-1-45-68-83-19; E-mail: tlambert{at}pasteur.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Livermore, D. M. (1992). Carbapenemases. Journal of Antimicrobial Chemotherapy 29, 609–13.[ISI][Medline]

2 . Chow, J. W. & Shlaes, D. M. (1991). Imipenem resistance associated with the loss of a 40 kDa outer membrane protein in Enterobacter aerogenes. Journal of Antimicrobial Chemotherapy 28, 499–504.[Abstract]

3 . De Champs, C., Henquell, C., Guelon, D., Sirot, D., Gazuy, N. & Sirot, J. (1993). Clinical and bacteriological study of nosocomial infections due to Enterobacter aerogenes resistant to imipenem. Journal of Clinical Microbiology 31, 123–7.[Abstract]

4 . Ehrhardt, A. F., Sanders, C. C., Thomson, K. S., Watanakunakorn, C. & Trujiliano-Martin, I. (1993). Emergence of resistance to imipenem in Enterobacter isolates masquerading as Klebsiella pneumoniae during therapy with imipenem/cilastatin. Clinical Infectious Diseases 17, 120–2.[ISI][Medline]

5 . Bush, K., Tanaka, S. K., Bonner, D. P. & Sykes, R. B. (1985). Resistance caused by decreased penetration of ß-lactam antibiotics into Enterobacter cloacae. Antimicrobial Agents and Chemotherapy 27, 555–60.[ISI][Medline]

6 . Lee, E. H., Nicolas, M. H., Kitzis, M. D., Pialoux, G., Collatz, E. & Gutmann, L. (1991). Association of two resistance mechanisms in a clinical isolate of Enterobacter cloacae with high-level resistance to imipenem. Antimicrobial Agents and Chemotherapy 35, 1093–8.[ISI][Medline]

7 . Raimondi, A., Traverso, A. & Nikaido, H. (1991). Imipenem- and meropenem-resistant mutants of Enterobacter cloacae and Proteus rettgerii lack porins. Antimicrobial Agents and Chemotherapy 35, 1174–80.[ISI][Medline]

8 . Mehtar, S., Tsakris, A. & Pitt, T. L. (1991). Imipenem resistance in Proteus mirabilis. Journal of Antimicrobial Chemotherapy 28, 612–5.[ISI][Medline]

9 . Mainardi, J.-L., Mugnier, P., Coutrot, A., Buu-Hoi, A., Collatz, E. & Gutmann, L. (1997). Carbapenem resistance in a clinical isolate of Citrobacter freundii. Antimicrobial Agents and Chemotherapy 41, 2352–4.[Abstract]

10 . Bradford, P. A., Urban, C., Mariano, N., Projan, S. J., Rahal, J. & Bush, K. (1997). Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC ß-lactamase, and the loss of an outer membrane protein. Antimicrobial Agents and Chemotherapy 41, 563–9.[Abstract]

11 . Mackenzie, F. M., Forbes, K. J., Dorai-John, T., Amyes, S. G. B. & Gould, I. M. (1997). Emergence of a carbapenem-resistant Klebsiella pneumoniae. Lancet 350, 783.[ISI][Medline]

12 . Report of the Comité de l'Antibiogramme de la Société Française de Microbiologie. (1996). Technical recommendations for in vitro susceptibility testing. Clinical Microbiology and Infection 2, Suppl. 1, 11–25.

13 . Willets, N. & Wilkins, B. (1984). Processing of plasmid DNA during bacterial conjugation. Clinical Microbiology Reviews 48, 21–41.

14 . Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

15 . Bush, K. & Singer, S. B. (1989). Effective cooling allows sonication to be used for liberation of ß-lactamases from Gram-negative bacteria. Journal of Antimicrobial Chemotherapy 24, 82–4.[ISI][Medline]

16 . Matthew, M., Harris, A. M., Marshall, M. J. & Ross, G. W. (1975). The use of analytical isoelectric focusing for detection and identification of ß-lactamases. Journal of General Microbiology 88, 169–78.[ISI][Medline]

17 . Verdet, C., Arlet, G., Ben Redjeb, S., Ben Hassen, A., Lagrange, P. H. & Philippon, A. (1998). Characterisation of CMY-4, an AmpC-type plasmid-mediated ß-lactamase in a Tunisian clinical isolate of Proteus mirabilis. FEMS Microbiology Letters 169, 235–40.[ISI][Medline]

18 . Takahashi, S. & Nagano, Y. (1984). Rapid procedure for isolation of plasmid DNA and application to epidemiological analysis. Journal of Clinical Microbiology 20, 608–13.[ISI][Medline]

19 . Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, USA 74, 5463–7.[Abstract]

20 . Diedrich, D. L., Summers, A. O. & Schnaitman, C. A. (1977). Outer membrane proteins of Escherichia coli. V. Evidence that protein I and bacteriophage-directed protein 2 are different polypeptides. Journal of Bacteriology 131, 598–607.[ISI][Medline]

21 . Shaw, K. J., Rather, P. N., Sabatelli, F., Mann, P., Munayyer, H., Mierzwa, R. et al. (1992). Characterization of the chromosomal aac(6')-Ic gene from Serratia marcescens. Antimicrobial Agents and Chemotherapy 36, 1447–55.[Abstract]

22 . Arakawa, Y., Ohta, M., Kido, N., Fujii, Y., Komatsu, T. & Kato, N. (1986). Close evolutionary relationship between the chromosomally encoded ß-lactamase gene of Klebsiella pneumoniae and the TEM ß-lactamase gene mediated by R plasmids. FEBS Letters 207, 69–74.[ISI][Medline]

23 . Martinez-Martinez, L., Pascual, A., Hernandez-Alles, S., Alvarez-Diaz, D., Suarez, A. I., Tran, J. et al. (1999). Roles of ß-lactamases and porins in activities of carbapenems and cephalosporins against Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 43, 1669–73.[Abstract/Free Full Text]

24 . Jacoby, G. A. (1994). Genetics of extended-spectrum ß-lactamases. European Journal of Clinical Microbiology and Infectious Diseases 13, Suppl. 1, S2–11.[Medline]

25 . Livermore, D. M. (1995). ß-Lactamases in laboratory and clinical resistance. Clinical Microbiology Reviews 8, 557–84.[Abstract]

26 . van de Klundert, J. A. M., van Gestel, M. H., Meerdink, G. & de Marie, S. (1988). Emergence of bacterial resistance to cefamandole in vivo due to outer membrane protein deficiency. European Journal of Clinical Microbiology and Infectious Diseases 7, 776–8.[ISI][Medline]

27 . Chen, H. Y. & Livermore, D. M. (1993). Activity of cefepime and other ß-lactam antibiotics against permeability mutants of Escherichia coli and Klebsiella pneumoniae. Journal of Antimicrobial Chemotherapy 32, Suppl. B, 63–74.[Abstract]

28 . Rice, L. B., Carias, L. L., Etter, L. & Shlaes, D. M. (1993). Resistance to cefoperazone–sulbactam in Klebsiella pneumoniae: evidence for enhanced resistance resulting from the coexistence of two different resistance mechanisms. Antimicrobial Agents and Chemotherapy 37, 1061–4.[Abstract]

29 . Martinez-Martinez, L., Hernandez-Alles, S., Alberti, S., Tomas, J. M., Benedi V. J. & Jacoby, G. A. (1996). In vivo selection of porin-deficient mutants of Klebsiella pneumoniae with increased resistance to cefoxitin and expanded-spectrum cephalosporins. Antimicrobial Agents and Chemotherapy 40, 342–8.[Abstract]

30 . Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103–19.[ISI][Medline]

31 . Bachmann, B. J. (1987). Derivations and genotypes of some mutant derivatives of Escherichia coli K-12. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Microbiology (Neidhartdt, F. C., Ingraham, J. L., Low, K. B., Magasanik, B., Schaechter, M. and Umbarger, H. E., Eds), Vol. 2., pp. 1190–219. American Society for Microbiology, Washington, DC.

32 . Spratt, B. G., Hedge, P. J., te Heesen, S., Edelman, A. & Broome-Smith, J. K. (1986). Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9. Gene 41, 337–42.[ISI][Medline]

Received 8 February 2000; returned 3 May 2000; revised 10 July 2000; accepted 24 August 2000