Identification of a chromosome-borne class C ß-lactamase from Erwinia rhapontici

Thierry Naas*, Daniel Aubert, Sophie Vimont and Patrice Nordmann

Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre Cedex, France

Received 17 August 2004; returned 27 August 2004; revised 1 September 2004; accepted 3 September 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Objectives: To characterize the ß-lactamase gene content of Erwinia rhapontici.

Methods: The ß-lactamase gene was cloned, sequenced and expressed in Escherichia coli.

Results: The cloned gene conferred a resistance pattern of an Ambler class C ß-lactamase in E. coli. The AmpC-type enzyme had a pI value of 8.6 and shared 62% amino acid sequence identity with that of Escherichia fergusonii. The ampC gene was associated with a regulatory ampR gene and ß-lactamase production was inducible.

Conclusions: This work provides further evidence of the molecular heterogeneity of ß-lactamases in Erwinia spp. and that plant-pathogenic enterobacterial species may constitute a reservoir of antibiotic resistance genes.

Keywords: AmpC , inducible , cephalosporinases , E. rhapontici


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Most members of the genus Erwinia generally cause disease to plants, vegetables and fruits.1 The phylogenetic relationship of the genus Erwinia with Enterobacteriaceae has been established based on 16S rDNA relatedness and it has been proposed that the genus Erwinia may be divided into four new genera, namely Erwinia, Pectobacterium, Pantoea and Brenneria.1 The genus Erwinia has been restricted to six species: Erwinia amylovora, Erwinia mallotivora, Erwinia persicina, Erwinia psidii, Erwinia rhapontici and Erwinia tracheiphila.1

Reports of Erwinia isolates as human pathogens are limited since most of the reported Erwinia isolates in clinical microbiology have now been assigned to the Pantoea group.2 The phytopathogen E. persicina has been isolated from human urinary tract infections2,3 and Pectobacterium chrysanthemi expresses virulence determinants that cause death of human gastrointestinal cells in culture.4 Detailed antibiotic resistance patterns of Erwinia species are known only for seven E. persicina strains3,4 and an expanded-spectrum Ambler class A ß-lactamase,5 ERP-1, has been characterized from E. persicina.3

The aim of this work was to characterize the ß-lactamase content of E. rhapontici. Cloning and sequencing of the ß-lactamase gene allowed us to compare its sequence with those of chromosomal- and plasmid-encoded ß-lactamases.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Bacterial strains and MIC determinations

E. rhapontici 105202T reference strain and representative reference strains of three groups of Erwinia sp.—E. persicina 105199T, E. mallotivora 105167T, E. psidii 105200T, E. amylovora 82.82T, Pectobacterium carotovorum subsp. carotovorum 82.83T, P. chrysanthemi 82.99T, Pectobacterium cacticida 105191T, Brenneria alni 104916T, Brenneria quercina 105201T, Brenneria rubrifaciens 105203T, Brenneria nigrifluens 105198T and Brenneria salicis 105204T—were from the Institut Pasteur (Institut Pasteur Strain Collection, Paris, France). Escherichia coli DH10B (Invitrogen, Cergy Pontoise, France) was used as the host strain for electroporation and cloning experiments. E. coli DH10B and E. coli JRG582, an ampDE deletion mutant, were used for ß-lactamase expression.6

Routine antibiograms, determined by the disc diffusion method, and MICs determined for selected ß-lactams by an agar dilution technique, were performed as previously reported,6 and interpreted according to the NCCLS guidelines.7

Cloning, plasmid content, mating-out assays and sequencing

Enzymes for DNA manipulation were from Amersham Biosciences (Orsay, France). Genomic DNAs of E. rhapontici 105202T were extracted as described previously.6 Plasmid DNAs of E. rhapontici and of recombinant E. coli DH10B clones were extracted using a Qiagen plasmid DNA maxi kit (Qiagen, Paris, France). Direct transfer of resistance marker into nalidixic acid-resistant E. coli DH10B was attempted by liquid and solid mating-out assays at 37°C, as previously reported.6

PCR amplifications were performed as previously described6 using the following laboratory designed primers: ERH-1A: 5'-AGTGGCGGTCCTGGTAAAAG-3', ERH-1B: 5'-AAGCATCTCCCAGCCAAGTC-3' that amplify a 736 bp internal fragment of ampC of E. rhapontici.

BamHI or Sau3AI partially restricted whole-cell DNAs were cloned into the BamHI-restricted pK19 vector carrying a kanamycin resistance marker, as described previously.8 Recombinant plasmids were transformed by electroporation (BioRad Gene Pulser II, Ivry-sur-Seine, France) into E. coli DH10B cells. E. coli harbouring recombinant plasmids were selected onto amoxicillin (50 mg/L) and kanamycin (50 mg/L)-containing trypticase soy agar plates.

Sequencing was performed using laboratory designed primers on an Applied Biosystem sequencer (ABI PRISM 3100). The nucleotide and the deduced protein sequences were analysed using software available on the internet (http://www.ncbi.nlm.nih.gov). The nucleotide sequence appears under GenBank accession number AY288518.

Biochemical techniques

Crude ß-lactamase extracts of E. rhapontici 105202T and of E. coli recombinant clones, total protein content measurement and analytical isoelectrofocusing (IEF) were performed as previously described.6 The specific ß-lactamase activity of the extracts was measured by UV spectrophotometry (ULTROSPEC 2000, Amersham Biosciences) as described previously with cefalothin as substrate.9 ß-Lactamase basal and induced levels (imipenem at 0.12 mg/L and cefoxitin at 2 mg/L) were determined as previously described.9 One unit of enzyme activity was defined as the activity that hydrolyses 1 µmoL of cefalothin or imipenem per min.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Cloning of ß-lactamase gene and susceptibility testing

Recombinant plasmids pERH-1, containing a 2.4 kb insert from the Sau3AI-cloning and pERH-2, containing a >15 kb insert from the BamHI-cloning were retained. IEF analysis of cultures of E. rhapontici 105202T and of recombinant E. coli clones had a single band of ß-lactamase activity with the same pI value of 8.6.

Plasmid detection, mating-out assays and electroporation attempts failed to detect plasmid DNA or a plasmid-encoded ß-lactam-resistance marker, suggesting a chromosomal location of the cloned genes.

The MICs of ß-lactams of E. rhapontici corresponded with that of an AmpC-type enzyme expressed at a low level (Table 1). E. coli DH10B (pERH-2) expressed a higher level of resistance including intermediate susceptibility to ceftazidime, cefotaxime and aztreonam. In all cases, susceptibility to imipenem, cefepime, cefpirome and moxalactam was observed. The inducibility of resistance to some ß-lactams, as evidenced on a routine antibiogram by an antagonism line between cefoxitin or imipenem and cefotaxime discs was not observed for E. rhapontici or E. coli transformants (data not shown).


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Table 1. MICs of ß-lactams for E. rhapontici reference strain 105202T, E. coli DH10B (pERH-1), E. coli DH10B (pERH-2) and E. coli DH10B reference strain

 
Identification of the ß-lactamase

Recombinant plasmid pERH-1 (Figure 1a) encoded a 1136-bp open reading frame (ORF) that shared significant sequence identity with those of chromosomal and plasmid-mediated class C ß-lactamase genes. Interestingly, this ORF starts with an unusual initiation codon ‘GTG’ and terminates with the three stop codons, ‘TAA-TAG-TGA’, lined up in the same frame, forming a tight translational stop and thus preventing any read-through transcript. Within the deduced amino acid sequence of the mature protein, an SXXK tetrad, characteristic of ß-lactamases possessing a serine active site, was found at positions 66–69.6,9,10 Three structural elements characteristic of AmpC ß-lactamases were also found:6,9,10 YAN at positions 152–154, DAES at positions 229–232 and KTG at positions 317–319.



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Figure 1. (a) Schematic representation of recombinant plasmids pERH-1 and pERH-2, containing the ampC coding region from E. rhapontici 105202T. The orientation of the genes is indicated with an arrow and solid lines denote the cloning vector. The double slash (//) represents regions of the inserts that have not been sequenced and that are not to scale. Homologies with protein sequences are indicated above or below open reading frames. (b) Antibiogram susceptibility test by disc diffusion showing a synergy image between cefoxitin (FOX) and cefalothin (CF) discs. Slight antagonism can be seen between cefalothin and imipenem (IMP).

 
The 379 amino acid-long AmpC type ß-lactamase was 62% and 61% identical to that of E. fergusonii9 and Morganella morganii,6 respectively, which were the closest AmpC enzymes. AmpC from E. rhapontici did not share sufficient sequence identity with known plasmid-mediated enzymes to be proposed as a progenitor. The LysR-type of the regulatory protein gene (ampR) was identified upstream of ampC (Figure 1a).11 This ORF started with a GTG codon and was transcribed in the opposite direction to that of the ampC gene. The predicted translation product of 292 amino acid residues aligned with the AmpR protein of M. morganii (68%).6 The 110 bp region between the ampR and ampC start codons contained putative overlapping promoters, as observed for most enterobacterial ampC/ampR genes.6,11

Downstream of the ampC gene, part of an ORF was found that coded for a putative Ybhb homologue. The partial protein sequence was 73% identical to the Ybhb protein of E. coli, which is thought to be a raf-1-like homologue in bacteria (GenBank accession number AF497476). In the upstream region, a partial ORF was found to encode a putative membrane protein that is 57% identical to that of Yersinia pestis (GenBank accession number AJ414159.1). Both genes, either upstream or downstream, are chromosomally encoded in that species, suggesting that the ampC/ampR genes are chromosomal as well. The genetic environment of this ampC-ER gene was different from that of other ampC genes described in Enterobacteriaceae, further underlining the heterogeniety of the genetic environment of these genes.6,9,10

Similar to E. coli, several enterobacterial species may remain susceptible to ß-lactams while harbouring non- or weakly expressed ß-lactamase genes. Therefore, several reference strains of Erwinia, Pectobacterium and Brenneria were screened by PCR using primers specific for ampC of E. rhapontici. Despite several attempts, no positive PCR results were obtained, even in less stringent conditions, indicating that similar ß-lactamase genes could not be identified from strains of the other Erwinia groups (data not shown).

Induction of ß-lactamase production

Basal ß-lactamase activities from cultures of E. rhapontici 105202T, E. coli DH10B (pERH-1), E. coli DH10B (pERH-2) and E. coli JRG582 (pERH-2) were 0.003, 0.39, 0.07 and 0.66 µmol/min/mg of protein, respectively. The absence of ampD in E. coli JRG582 led to a 10-fold increase in ß-lactamase expression as compared with E. coli DH10B (pERH-2). Induction of the ß-lactamase expression using cefoxitin or imipenem as inducers with cultures of E. rhapontici 105202T and of E. coli (pERH-2) was 75- and 3.5-fold, respectively. No induction was seen with E. coli (pERH-1), which is consistent with the deletion of one third of an ampR gene in plasmid pERH-1; no induction was seen either with E. coli JRG582 (pERH-2), for which a constitutive expression has been observed, consistent with what is observed in other systems in the absence of the ampD gene.6 E. coli DH10B (pERH-2) had a basal ß-lactamase expression level five-fold lower than that of E. coli DH10B (pERH-1). Thus, AmpR acts as a moderate repressor of cephalosporinase expression in the absence of a ß-lactam inducer and as a strong activator in its presence.

Lack of an antagonism image on a routine antibiogram may be falsely taken as an absence of inducibility of ß-lactamase expression (Figure 1b). It is only when cefalothin and imipenem discs are in close contact that a very slight antagonism is seen (Figure 1b). Instead of an antagonism, a synergy image between cefalothin and cefoxitin was observed (Figure 1b). This cefoxitin-inhibitory property, which has been reported for some chromosome-borne cephalosporinases of Citrobacter freundii, Providencia stuartii and Hafnia alvei,10 may explain why E. coli DH10B (pERH-1/2) remained fully susceptible to cefoxitin. Hydrolysis experiments revealed that culture extracts expressing AmpC-ER had no detectable specific hydrolysis activity against cefoxitin, but were strongly inhibited by this substrate (IC50=0.05 µM).

Conclusion

This work identified the first chromosomally encoded and inducible class C ß-lactamase from a phytopathogenic enterobacterial species, which is a natural contaminant and pathogen of fruits, plants and vegetables. It underlines further that class C ß-lactamases may be identified in enterobacterial species other than those involved in human infections. Furthermore, this work provides evidence that plant-pathogenic Enterobacteriaceae may constitute a reservoir of antibiotic resistance genes, as exemplified by Tn5393, a transposon from E. amylovora, which carries the streptomycin resistance operon and which is now identified in plant, animal and human bacterial isolates.12


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
This work was funded by a grant from the Ministère de la Recherche (grant UPRES-EA 3539), Université Paris XI, Paris France and the European Community (6th PCRD, LSHM-CT-2003–503–335).


    Footnotes
 
* Corresponding author. Tel: +33-1-45-21-29-86; Fax: +33-1-45-21-63-40; Email: thierry.naas{at}bct.ap-hop-paris.fr


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
1 . Kwon, S. W., Go, S. J., Kang, H. et al. (1997). Phylogenetic analysis of Erwinia species based on 16S rRNA gene sequences. International Journal of Systematic Bacteriology 47, 1061–7.[Abstract/Free Full Text]

2 . O'Hara, C. M., Steigerwalt, A. G., Hill, B. C. et al. (1998). First report of a human isolate of Erwinia persicinus. Journal of Clinical Microbiology 36, 248–50.[Abstract/Free Full Text]

3 . Vimont, S., Poirel, L., Naas, T. et al. (2002). Identification of a chromosome-borne expanded-spectrum class A ß-lactamase from Erwinia persicina. Antimicrobial Agents and Chemotherapy 46, 3401–5.[Abstract/Free Full Text]

4 . Duarté, X., Anderson, C. T., Grimson, M. et al. (2001). Erwinia chrysanthemi strains cause death of human gastrointestinal cells in culture and express an intimin-like protein. FEMS Microbiology Letters 190, 81–6.[CrossRef][ISI]

5 . Ambler, R. P., Coulson, A. F., Frère, J.-M. et al. (1991). A standard numbering scheme for the class A ß-lactamases. Biochemical Journal 276, 269–70.[ISI][Medline]

6 . Poirel, L., Guibert, M., Girlich, D. et al. (1999). Cloning, sequence analyses, expression, and distribution of ampC-ampR from Morganella morganii clinical isolates. Antimicrobial Agents and Chemotherapy 43, 769–76.[Abstract/Free Full Text]

7 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Sixth Edition: Approved Standard M7-A6. NCCLS, Villanova, PA, USA.

8 . Pridmore, R. D. (1987). New and versatile cloning vectors with kanamycin resistance marker. Gene 56, 309–12.[CrossRef][ISI][Medline]

9 . Naas, T., Aubert, D., Fortineau, N. et al. (2002). Cloning and sequencing of the ß-lactamase gene and surrounding DNA sequences of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii, Escherichia fergusonii and Enterobacter cancerogenus. FEMS Microbiology Letters 215, 81–7.[CrossRef][ISI][Medline]

10 . Girlich, D., Naas, T., Bellais, S. et al. (2000). Biochemical-genetic characterization and regulation of expression of an ACC-1-like chromosome-borne cephalosporinase from Hafnia alvei. Antimicrobial Agents and Chemotherapy 44, 1470–8.[Abstract/Free Full Text]

11 . Hanson, N. D. & Sanders, C. C. (1999). Regulation of inducible AmpC ß-lactamase expression among Enterobactericeae. Current Pharmaceutical Design 5, 881–94.[ISI][Medline]

12 . Sundin, G. W. (2002). Distinct recent lineages of the strA-strB streptomycin-resistance genes in clinical and environmental bacteria. Current Microbiology 45, 63–9.[CrossRef][ISI][Medline]





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