Characterization, cloning and sequence analysis of the inducible Ochrobactrum anthropi AmpC ß-lactamase

Catherine S. Higgins, Matthew B. Avison*,, Lee Jamieson, Alan M. Simm, Peter M. Bennett and Timothy R. Walsh

Bristol Centre for Antimicrobial Research and Evaluation, Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ochrobactrum anthropi is resistant to most cephalosporins and penicillins due, at least in part, to the inducible expression of a single ß-lactamase. The ß-lactamase gene has been cloned and sequenced. It encodes an AmpC-type class 1 serine active-site enzyme that hydrolyses mainly cephalosporins and is resistant to inhibition by clavulanic acid. Expression of the ampC gene is inducible via a typical AmpR regulator, which is encoded upstream of ampC. Inducible expression is retained following cloning of O. anthropi ampRampC into Escherichia coli, confirming that the signal for AmpR activation in O. anthropi is the same as that used in the Enterobacteriaceae. This is the first reported example of an AmpC ß-lactamase outside of the {gamma}-subdivision of the bacterial kingdom. Genomic searches of other non-{gamma}-subdivision bacteria revealed a homologous ampRampC cluster in the plant symbiont, Sinorhizobium meliloti.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ochrobactrum anthropi is a non-fastidious, non-fermenting, Gram-negative cocco-bacillus.1 It is an emerging nosocomial pathogen, which has been found in environmental and hospital water sources.14 It has been reported to cause bacteraemias in immunocompromised patients, particularly oncology patients with indwelling catheters.2,4 Occasionally, probably because of its ability to survive in water supplies, epidemic outbreaks occur.4

Initially categorized as a member of the genus Achromobacter, DNA–DNA hybridization and 16S sequencing data led to the foundation of the genus Ochrobactrum in 1988 and its partitioning within the Rhizobiaceae group. This group includes plant and animal intracellular pathogens such as Agrobacterium spp., Bartonella spp. and Brucella spp.5,6 Among all these bacteria, O. anthropi is most closely related to the Brucellaceae, and recent reports have described immunological cross-reactivity between Brucella spp. and O. anthropi antigens.7,8 However, because O. anthropi constitutes a heterogeneous group of bacteria on the basis of phenotypic characterization and DNA–DNA hybridization studies, further subdivision of the genus into two species, O. anthropi and Ochrobactrum intermedium has recently been proposed, with O. intermedium being more closely related to Brucella spp.9

In general, O. anthropi strains are reported to be resistant to cephalosporins and penicillins, but susceptible to carbapenems, quinolones and tetracyclines.2,4 Nothing has been reported previously concerning the mechanism of ß-lactam resistance in O. anthropi. The O. anthropi isolates we have studied come from three oncology patients with bacteraemias that recurred following courses of piperacillin/tazobactam and ceftazidime therapy. The antimicrobial susceptibilities of the isolates were determined, and the production of ß-lactamase was investigated as a possible cause of ß-lactam resistance.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and plasmids

Clinical isolates of O. anthropi were recovered over a period of several years from blood cultures of bacteraemic oncology patients being treated at the Bristol Royal Infirmary (BRI). Isolates were plated on to nutrient agar (Oxoid, Basingstoke, UK) to check culture purity and the identity of each was confirmed using API 20NE test strips (bioMérieux, La Balme les Grottes, France). Bacteria were grown at 37°C in air, unless otherwise stated. Bacterial strains and plasmids used in this study are listed in Table IGo.


View this table:
[in this window]
[in a new window]
 
Table I.  The bacterial strains and plasmids used in this study
 
Materials

Unless otherwise stated, media used were nutrient broth and nutrient agar (Oxoid). ß-Lactams used were nitrocefin (Beckton Dickinson, Cockeysville, MD, USA); clavulanic acid and BRL 42715 (SmithKline Beecham, Worthing, UK); ampicillin and cephaloridine (Sigma Chemical Co., St Louis, MO, USA); ceftazidime (Glaxo Laboratories Ltd, Greenford, UK); piperacillin (Lederle, Carolina, Puerto Rico); and meropenem (Zeneca Pharmaceuticals, Macclesfield, UK). PCR primers were purchased from Sigma Genosys Ltd (Pampisford, UK). General reagents for DNA manipulation were obtained from Gibco-BRL (Life Technology Ltd, Paisley, UK). All other general reagents were from Sigma Chemical Co. or BDH, both of Poole, Dorset, UK.

Susceptibility tests

Antibiotic susceptibility was determined by Etest (AB Biodisk, Solna, Sweden) on IsoSensitest agar (Oxoid) with an inoculum of 0.5 McFarland. The MIC of the test antibiotic was defined as the lowest concentration of the antibiotic that prevented growth after incubation at 37°C for 24 h.

Induction of ß-lactamase expression, isolation and assay of ß-lactamases

Induction of ß-lactamase expression was attempted and crude cell extracts were prepared as described previously.10 Hydrolysis of ß-lactam antibiotics was examined by spectrophotometric analysis as described previously10 at the following wavelengths: 233, 265, 265, 299, 482 and 233 nm, and using the following extinction coefficients: 809, 6980, 9000, 2500, 17400 and 936 Au/M/cm, respectively, for ampicillin, cephaloridine, ceftazidime, meropenem, nitrocefin and piperacillin. The protein concentration of each bacterial extract was determined using the Bio-Rad protein assay reagent (Bio-Rad, München, Germany) according to the manufacturer's instructions. One unit of ß-lactamase activity was defined as that required to hydrolyse 1 nmol of substrate per min at 25°C. Specific activity was defined as the number of units of ß-lactamase per mg of protein in the cell extract.

Isoelectric focusing

Ten micrograms of total protein from each bacterial extract (above) were resolved by isoelectric focusing (IEF) gel electrophoresis, and ß-lactamase(s) was visualized as described previously.11

Cloning the O. anthropi ampR–ampC locus

Chromosomal DNA was prepared from O. anthropi N217I and general molecular biology techniques were performed as described previously.12 DNA was restricted with EcoRI, ligated into appropriately restricted pSU1813 and used to transform Escherichia coli strain UB5252,10 by electrotransformation with a Gene Pulser (Bio-Rad) set at 2.5 V, 25 µF and 200 {Omega}. UB5252 is a permeability mutant of E. coli HfrH, which is hypo-susceptible to ß-lactams, allowing easier cloning of ß-lactamase genes that are poorly expressed in E. coli. Transformed bacteria were selected on agar containing chloramphenicol and cephaloridine (both at 30 mg/L). Resistant transformants were recovered, plasmid DNA was isolated and the insert was sequenced using an ABI PRISM 377 automated DNA sequencer. Sequences were determined on both strands using a custom primer walking strategy.12

Polymerase chain reaction

Genomic polymerase chain reaction (PCR) was performed as described previously.14 The primers used to amplify O. anthropi ampC were (forward) 5'-GAGATGCGGTCGAACCAT-3' and (reverse) 5'-CTGACTGAACTGCTGCCG-3'. The primers were designed to span the entire coding sequence of the ampC gene. PCR products were purified using a QIAquick PCR purification kit (Qiagen Ltd, Crawley, UK) according to the manufacturer's instructions and sequenced on both strands using the PCR primers to initiate sequencing.

In silico sequence analysis

Computer-assisted sequence manipulation and basic alignments were done with the Lasergene software package (DNA star, Madison, WI). Interrogation of the NCBI microbial BLAST database with O. anthropi AmpC protein sequence was carried out using the tblastn search tool.15 Positive M13 random contigs (average size c. 600 bp) from the Sinorhizobium meliloti genome database were aligned in Lasergene using Seqman to produce a contiguous sequence for the ampR and ampC genes. Sequence data for S. meliloti were generated by S. R. Long and her colleagues at the Department of Biological Sciences, Howard Hughes Medical Institute and the Stanford DNA Sequencing and Technology Centre.

For the phylogenetic analysis, an alignment of published AmpC protein sequences was produced using the CLUSTAL W program16 applying the BLOSUM matrix with a gap-opening penalty of 10 and a gap-extension penalty of 0.1. The resulting alignment was analysed using a maximum likelihood method17 with the program protml18 and the Jones, Taylor, Thornton substitution model.19


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
ß-Lactam resistance profile and expression of ß-lactamases by O. anthropi

Initially, the MICs of a variety of ß-lactams for the three O. anthropi clinical isolates were determined. The three isolates were resistant (according to BSAC breakpoints for the Enterobacteriaceae20) to all cephalosporins and penicillins tested (including four combinations of ß-lactam plus clavulanic acid or tazobactam), and aztreonam, but were susceptible to carbapenems (Table IIGo).


View this table:
[in this window]
[in a new window]
 
Table II.  MICs of various antibiotics for the O. anthropi isolates
 
Upon finding this resistance profile, we suspected the production of at least one ß-lactamase. Expression of ß-lactamase activity was confirmed by the ability of cell extracts from isolate N217I to hydrolyse nitrocefin. Specific rates of hydrolysis were very low (c. 20 U/mg), and to determine whether the ß-lactamase activity was inducible, isolate N217I was challenged with cefoxitin (10 mg/L, 2 h). Using nitrocefin as a substrate, cell extracts of induced N217I had about 50-fold more activity (1044 U/mg) than extracts from uninduced cultures.

IEF using induced cell extracts revealed only one ß-lactamase band (pI = 9.0) following nitrocefin staining (data not shown). The enzyme appeared to be a strict cephalosporinase, as induced cell extracts hydrolysed nitrocefin, cephaloridine and ceftazidime, but not meropenem, ampicillin or piperacillin (Table IIIGo). The ß-lactamase activity (nitrocefin hydrolysis) was completely inhibited by the broad-spectrum serine ß-lactamase inhibitor, BRL 42715 (10 µM), but not by clavulanic acid (10 µM) or EDTA (50 mM) (Table IIIGo). Almost identical results were obtained when analysing the other two clinical O. anthropi strains (data not shown).


View this table:
[in this window]
[in a new window]
 
Table III.  Substrate profile of the ß-lactamase activity found in extracts of induced O. anthropi isolate N217I
 
Cloning the O. anthropi ß-lactamase gene, ampC, and regulatory gene, ampR

The ß-lactamase gene from O. anthropi isolate N217I was cloned into E. coli UB5252 following the construction of an EcoRI genebank of N217I genomic DNA using cloning vector pSU18. Recombinants were selected with chloramphenicol and cephaloridine (both at 30 mg/L) and all contained pSU18 carrying a 4.8 kb insert. One such plasmid recombinant was designated pUB6052. E. coli UB5252 containing pUB6052 was found to have decreased susceptibility to cefotaxime and ceftazidime, but not to ampicillin or meropenem, when compared with untransformed UB5252 (Table IVGo). Extracts of UB5252:pUB6052 hydrolysed cephaloridine, but not meropenem or ampicillin (Table IVGo). Finally, IEF analysis showed the cloned ß- lactamase had a pI of 9.0, indistinguishable from that expressed by O. anthropi strain N217I (data not shown).


View this table:
[in this window]
[in a new window]
 
Table IV.  Resistance profile and ß-lactamase activity of E. coli recombinants containing pUB6052
 
Having cloned a ß-lactamase gene encoding an enzyme with identical properties to those of the O. anthropi N217I ß-lactamase, the pUB6052 insert was sequenced. The sequence (EMBL accession number AJ299421) revealed an ampC-type ß-lactamase gene downstream of, and oppositely oriented to, a typical ampR regulator gene21 (Figure 1Go).



View larger version (56K):
[in this window]
[in a new window]
 
Figure 1.  The nucleotide and deduced amino acid sequence of O. anthropi ampRampC. The putative ribosome binding sites are double underlined and the putative rho-independent transcriptional terminators are single underlined. The amino acids involved in signal peptide cleavage of AmpC are bold, and the cleavage site is marked with an asterisk.

 
Sequence analysis of ampC from O. anthropi

The O. anthropi N217I ampC gene extends for 1173 nucleotides, encoding a polypeptide of 390 amino acids. A credible ribosome binding site is located 15 nucleotides upstream from the initiation codon, and 16 nucleotides downstream of the gene there is an inverted repeat followed by a poly(T) run, which is likely to be a Rho-independent transcription terminator.22 No obvious promoter with significant homology to the {sigma}70 promoter of E. coli has been identified for ampC, though there are a number of excellent putative –10 boxes (Figure 1Go).23 A credible 25 amino acid secretion leader peptide24 in AmpC is defined by alanine residues at positions 25 and 26. The mature 365 amino acid protein has a predicted molecular weight of 39.6 kDa and a predicted pI of 9.0 (the same as that determined experimentally).

PCR primers were designed from the N217I ampC sequence and used to amplify the gene from the other two clinical O. anthropi isolates. The PCR amplicons were sequenced to assess the level of sequence heterogeneity amongst the O. anthropi ampC genes. In fact, all three sequences were identical. Importantly, this also demonstrates that the cloned ß-lactamase gene is from O. anthropi.

Analysis of O. anthropi AmpR and the mechanism of ampC induction

The 193 bp ampRampC intercistronic region contains a putative –10 box for ampR, but no recognizable –35 box (Figure 1Go).23 Having shown that the O. anthropi ampC is inducible, we measured the inducibility of an E. coli construct containing pUB6052 (ampRampC). Following cefoxitin challenge at sub-MIC levels, an increase in ß- lactamase activity was observed (Table IVGo) in extracts of E. coli carrying pUB6052. In addition, cloning of pUB6052 into the ampD mutant, JRG582 (which has high basal levels of the enterobacterial AmpR signal25) resulted in significantly higher ß-lactamase activity than those in HfrH,26 the parent of JRG582,25 containing pUB6052 (Table IVGo).

The presence of AmpC ß-lactamases in other members of the {alpha}-subdivision

The O. anthropi AmpC represents the first AmpC ß- lactamase to be found outside of the {gamma}-subdivision of the bacterial kingdom. We used the O. anthropi AmpC amino acid sequence to search for homologues in genome sequences from bacteria that are not part of the {gamma}-subdivision. Only one homologue was found, in S. meliloti. In this case, the ampC gene is also downstream of an ampR homologue (data not shown). We have not determined whether the S. meliloti ampC gene encodes an active ß-lactamase, or if its expression is inducible. The two novel AmpC amino acid sequences were aligned with all previously published examples of chromosomal AmpC-type ß-lactamases (Figure 2Go). This information was used to construct a phylogenetic tree for chromosomal AmpC enzymes (Figure 3Go).




View larger version (258K):
[in this window]
[in a new window]
 
Figure 2.  Alignment of published chromosomal AmpC amino acid sequences. Multiple alignment of AmpC proteins as described in Materials and methods. Regions of identity are shaded in black and regions of similarity are shaded grey. EMBL accession numbers for the published AmpC sequences are: CAA56561, Aeromonas veronii bv Sobia; Q9L3I5, Aeromonas hydrophila; Q44219, Aeromonas jandaei; S13408, Pseudomonas aeruginosa; P05193, Citrobacter freundii; P00811, E. coli; P05364, Enterobacter cloacae; P45460, Yersinia enterocolitica; Q9K351, Hafnia alvei; P18539, Serratia marcescens; Q48743, Lysobacter lactamgenus; O05465, Psychrobacter immobilis; Q9L4R5, Acinetobacter baumannii.

 


View larger version (24K):
[in this window]
[in a new window]
 
Figure 3.  Phylogenetic tree based on a comparison of published AmpC amino acid sequences. The alignment shown in Figure 2Go was analysed as described in Materials and methods to produce a phylogenetic tree of the AmpC amino acid sequences (a). Each number represents a node of sequence divergence and the length of the branch represents the estimated number of substitutions per 100 amino acids along the branch leading to each node. The raw data for divergence to each node are shown in (b). The ‘L’ value, representing the likelihood that this tree is the best description of the aligned sequences is –9545 ± 226.17 This is an unrooted tree.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The data presented in our report clearly demonstrate the presence of an ampR–ampC locus in the {alpha}-subdivision bacterium, O. anthropi, which is related to those commonly found in members of the {gamma}-subdivision of the bacterial kingdom.27 The ß-lactamase encoded by O. anthropi ampC is inducible and is a typical member of the class, namely, a cephalosporinase with a pI of 9.0. The ampR–ampC gene arrangement, adjacent genes that are divergently transcribed from a common intergenic region, is also typical of that seen in several members of the {gamma}-subdivision.27 That the mechanism of ampC induction in O. anthropi is essentially the same as that described for the ampC genes of Citrobacter freundii and Enterobacter cloacae was demonstrated by the finding that AmpC expression was constitutive and high level when ampR–ampC were introduced into an E. coli ampD mutant, and inducible upon ß-lactam challenge when introduced into an ampD+ E. coli strain. Hence, production of the O. anthropi AmpC ß- lactamase is coupled to peptidoglycan recycling, as in the Enterobacteriaceae.21

The multiple alignment of class C AmpC ß-lactamases shown in Figure 2Go reveals a large degree of sequence heterogeneity. This variation has come about through divergent evolution, and the extent of this variation implies that the ancestral ampC gene was acquired a considerable time ago. There are regions within the AmpC proteins with a high degree of sequence conservation, however, and upon mapping these conserved regions on to known AmpC crystal structures,28 all can be associated with recognizable structural motifs within the enzyme, so a high degree of homology is unsurprising. There is likely to be strong selective pressure on these proteins to maintain the integrity of the active site and hence the individual motifs stabilizing that site. Other, less important, regions change more freely, however.

The phylogenetic analysis (Figure 3Go) of the AmpC proteins clearly places O. anthropi AmpC within the same group as the predicted AmpC from another {alpha}-proteobacterium, S. meliloti. As this analysis produces an unrooted tree (Figure 3Go), it is not possible to say with certainty what the ancestral AmpC sequence was. More AmpC sequence data from organisms outside the {gamma}-subdivision will help clarify this situation. However, our current study does indicate that AmpC enzymes evolved before the divergence of the {alpha} and {gamma} groups, which is more evidence that the acquisition of ampC is an ancient event, and occurred millennia before the first clinical use of ß-lactams.

Whilst the {gamma}-subdivision of the bacterial kingdom contains most of the common animal pathogens, the {alpha}- subdivision is composed mainly of environmental organisms and plant symbionts such as S. meliloti. Clearly, in these latter organisms, the selective pressure of clinical ß-lactam use has not maintained the presence of AmpC. The true role of AmpC and other ß-lactamases is unknown, though they could have evolved to protect cells against ß-lactams in the environment. It is just as likely that AmpC enzymes serve a useful physiological purpose, however. That production of AmpC is inducible argues that the enzyme is useful to the cell during specific circumstances, rather than all the time. It was recently reported that the AmpC ß-lactamase of E. cloacae displays d,d-carboxypeptidase activity, suggestive of a role in peptidoglycan metabolism.29 This correlates well with the AmpC induction process being associated with peptidoglycan turnover.21

The greatest problem of clinical antibiotic resistance is caused by acquired ß-lactamases, usually because these are expressed at high levels.30 In contrast, inducible ß-lactamases rarely cause clinical resistance, though mutations that cause ß-lactamase overexpression can lead to therapeutic failure.30 It would seem strange to evolve a sophisticated induction mechanism to bring about ß-lactam resistance, if the only way resistance occurs is following mutation of the induction machinery. Clearly, however, the potential to produce any ß-lactamase is an advantage for a pathogen that comes into contact with ß-lactams. O. anthropi is such an organism, and it may be that its intrinsic antibiotic resistance mechanisms are part of the reason for its recent rise. O. anthropi is categorized amongst the Brucellaceae, and antigenic cross-reactivity between O. anthropi and Brucella abortus has been noted.79 Interestingly, a recent report showed that Brucella spp. are resistant to cephalosporins during growth in nutrient broth or on nutrient agar.31 It is therefore possible that these organisms also encode an ampC ß-lactamase.


    Acknowledgments
 
We thank the British Society for Antimicrobial Chemotherapy for continued group support; Dr Emma Williamson, BRI, Bristol, UK, for donating the clinical isolates used; Rachel Horton (funded by a BSAC vacation scholarship) for technical assistance, and Jennie Douthwaite, Department of Biochemistry, University of Bristol for DNA sequencing. This work was funded by grants from the Wellcome Trust. C. S. H. was in receipt of a Biotechnology and Biological Sciences Research Council CASE studentship in collaboration with GlaxoSmithKline Pharmaceuticals.


    Notes
 
* Correspondence address. Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK. Tel: +44-117-9287439; Fax: +44-117-9288274; E-mail: Matthewb.Avison{at}bris.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 .  Holmes, B., Popoff, M., Kiredjian, M. & Kersters, K. (1988). Ochrobactrum anthropi gen. nov., sp. nov. from human clinical specimens and previously known as group Vd. International Journal of Systematic Bacteriology 38, 406–16.

2 .  Gransden, W. R. & Eykyn, S. J. (1992). Seven cases of bacteraemia due to Ochrobactrum anthropi. Clinical Infectious Diseases 15, 1068–9.[ISI][Medline]

3 .  Babic, I., Fischer-Le Saux, M., Giraud, E. & Boemare, N. (2000). Occurrence of natural dixenic associations between the symbiont Photorhabdus luminescens and bacteria related to Ochrobactrum spp. in tropical entomopathogenic Heterorhabditis spp. Microbiology 146, 709–18.[Abstract/Free Full Text]

4 .  Deliere, E., Vu-Thien, H., Levy, V., Barquins, S., Schlegel, L. & Bouvet, A. (2000). Epidemiological investigation of Ochrobactrum anthropi strains isolated from a haematology unit. Journal of Hospital Infection 44, 173–8.[ISI][Medline]

5 .  Yanagi, M. & Yamasato, K. (1993). Phylogenetic analysis of the family Rhizobiaceae and related bacteria by sequencing of 16S rRNA gene using PCR and DNA sequencing. FEMS Microbiology Letters 107, 115–20.[ISI][Medline]

6 .  Moreno, E. (1998). Genome evolution within the {alpha}-Proteobacteria: why do some bacteria not possess plasmids and others exhibit more than one different chromosome? FEMS Microbiology Reviews 22, 255–75.[ISI][Medline]

7 .  Velasco, J., Diaz, R., Grillo J., Barberan, M., Marin, C., Blasco, J. M. & Moriyon, I. (1997). Antibody and delayed-type hypersensitivity responses to Ochrobactrum anthropi cytosolic and outer membrane antigens in infections by smooth and rough Brucella spp. Clinical and Diagnostic Laboratory Immunology 4, 279–84.[Abstract]

8 .  Cloeckaert, A., Tibor, A. & Zygmunt, M. S. (1999). Brucella outer membrane lipoproteins share antigenic determinants with bacteria of the family Rhizobiaceae. Clinical and Diagnostic Laboratory Immunology 6, 627–9.[Abstract/Free Full Text]

9 .  Velasco, J., Romero, C., Lopez-Goni, I., Leiva, J., Diaz, R. & Moriyon, I. (1998). Evaluation of the relatedness of Brucella spp. and Ochrobactrum anthropi and description of Ochrobactrum intermedium sp. nov., a new species. International Journal of Systematic Bacteriology 48, 759–68.[Abstract/Free Full Text]

10 . Avison, M. B., Niumsup, P., Walsh, T. R. & Bennett P. M. (2000). Aeromonas hydrophila AmpH and CepH ß-lactamases: derepressed expression in mutants of Escherichia coli lacking creB. Journal of Antimicrobial Chemotherapy 46, 695–702.[Abstract/Free Full Text]

11 . Avison, M. B., Bennett, P. M. & Walsh, T. R. (2000). ß-Lactamase expression in Plesiomonas shigelloides. Journal of Antimicrobial Chemotherapy 45, 877–80.[Abstract/Free Full Text]

12 . Sambrook, J., Fritsch, E. F. & Maniatis, T. (Eds) (1989). Strategies for cloning in plasmid vectors. In Molecular Cloning: A Laboratory Manual, 2nd edn, pp. 53–104. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

13 . Martinez, E., Bartolome, B. & de la Cruz, F. (1988). pACYC184-derived cloning vectors containing the multiple cloning site and lacZ-alpha reporter gene of pUC8/9 and pUC18/19. Gene 68, 159–62.[ISI][Medline]

14 . Avison, M. B., von Heldreich, C. J., Higgins, C. S., Bennett, P. M. & Walsh, T. R. (2000). A TEM ß-lactamase encoded on an active Tn1-like transposon in the genome of a clinical isolate of Stenotrophomonas maltophilia. Journal of Antimicrobial Chemotherapy 46, 879–84.[Abstract/Free Full Text]

15 . Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389–402.[Abstract/Free Full Text]

16 . Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673–80.[Abstract]

17 . Kishino, H. & Hsaegawa, M. (1989). Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in Hominoidea. Journal of Molecular Evolution 29, 170–9.[ISI][Medline]

18 . Adachi, J. & Hasegawa, M. (1992). Amino acid substitution of proteins coded for in mitochondrial DNA during mammalian evolution. Japanese Journal of Genetics 67, 187–97.[ISI][Medline]

19 . Jones, D. T., Taylor, W. R. & Thornton, J. M. (1992). The rapid generation of mutation data matrices from protein sequences. Computer Applications in the Biosciences 8, 275–82.[Abstract]

20 . Anonymous. (1988). Breakpoints in in-vitro antibiotic sensitivity testing. Report by a working party of the British Society for Antimicrobial Chemotherapy. Journal of Antimicrobial Chemotherapy 21, 701–10.[ISI][Medline]

21 . Weidemann, B., Dietz, B. & Pfeifle, D. (1998). Induction of ß-lactamases in Enterobacter cloacae. Clinical Infectious Diseases 27, S42–7.[ISI][Medline]

22 . Yagar, T. D. & von Hippel, P. H. (1996). Transcriptional elongation and termination in Escherichia coli. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, Vol. 1, 2nd edn, (Neidhardt, F. C., Curtiss, R., III, Ingram, J. L., Lin, E. C. C., Low, K. B., Magasanik, B. et al., Eds), pp. 1241–75. ASM Press, Washington, DC.

23 . Record, M. T., Reznikoff, W. S., Craig, M. L., McQuande, K. L. & Schlax, P. J. (1996). Escherichia coli RNA polymerase (E{sigma}70), promoters and the kinetics of the steps of transcription initiation. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, Vol. 1, 2nd edn, (Neidhardt, F. C., Curtiss, R., III, Ingram, J. L., Lin, E. C. C., Low, K. B., Magasanik, B. et al., Eds), pp. 792–821. ASM Press, Washington, DC.

24 . Von Heijne, G. (1983). Patterns of amino acids near signal-sequence cleavage sites. European Journal of Biochemistry 133, 17–21.[Abstract]

25 . Lindquist, S., Galleni, M., Lindberg, F. & Normark, S. (1989). Signalling proteins in enterobacterial AmpC ß-lactamase regulation. Molecular Microbiology 3, 1121–30.[ISI][Medline]

26 . Low, K. B. (1973). Escherichia coli K-12 F-prime factors, old and new. Bacteriological Reviews 36, 587–607.[ISI]

27 . Bush, K., Jacoby, G. A. & Medeiros, A. A. (1995). A functional classification scheme for ß-lactamases and its correlation with molecular structure. Antimicrobial Agents and Chemotherapy 39, 1211–33.[Free Full Text]

28 . Lobkovsky, E., Moews, P. C., Liu, H., Zhao, H., Frere, J.-M. & Knox, J. R. (1993). Evolution of an enzyme activity: crystallographic structure at 2-Å resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase. Proceedings of the National Academy of Sciences, USA 90, 11257–61.[Abstract]

29 . Morisini, M. I., Ayala, J. A., Baquero, F., Matinez, J. L. & Blázquez, J. (2000). Biological cost of AmpC production for Salmonella enterica serotype Typhimurium. Antimicrobial Agents and Chemotherapy 44, 3137–43.[Abstract/Free Full Text]

30 . Nordmann, P. (1997). Trends in ß-lactam resistance among Enterobacteriaceae. Clinical Infectious Diseases 27, S100–6.[ISI]

31 . Rolain, J.-M., Maurin, M. & Raoult, D. (2000). Bactericidal effect of antibiotics on Bartonella and Brucella spp.: clinical implications. Journal of Antimicrobial Chemotherapy 46, 811–4.[Abstract/Free Full Text]

Received 18 December 2000; returned 22 February 2001; revised 1 March 2001; accepted 2 March 2001