JAC Advance Access originally published online on June 27, 2006
Journal of Antimicrobial Chemotherapy 2006 58(3):661-664; doi:10.1093/jac/dkl267
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Characterization of defective ß-lactamase genes in Yersinia enterocolitica
1 Departamento de Biología Molecular, Facultad de Medicina, Universidad de Cantabria Santander, Spain 2 Servicio de Microbiología, Hospital Universitario Marqués de Valdecilla Santander, Spain
*Corresponding author. Tel: +34-942-201948; Fax +34-942-201945; E-mail: jmglobo{at}unican.es
Received 7 April 2006; returned 3 May 2006; revised 8 May 2006; accepted 1 June 2006
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Abstract |
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Objectives: To study at the molecular level the heterogeneity of expression of the two chromosomal ß-lactamases, BlaA and BlaB, in Yersinia enterocolitica strains isolated from clinical samples.
Methods: MIC determination by the agar dilution method and ß-lactamase assays was performed to determine the resistance level conferred by these enzymes. DNA cloning, PCR and direct sequencing were used to detect the presence of mutations.
Results: The blaA allele from strain IP97 (blaA97) was found to carry a deletion of 51 bp which entirely abolished its ß-lactamase activity. Both the ampR gene and the promoter region of strain Y56 were shown to be functional by a gene swapping experiment. The blaB allele from strain Y56 was found to carry two point mutations, only one of them resulting in a change in the amino acid sequence of the protein. This single amino acid change created a practically inactive BlaB or AmpC cephalosporinase in Y. enterocolitica Y56.
Conclusions: The lack of activity observed in the ß-lactamases of some Y. enterocolitica isolates was due to the presence of point mutations or small deletions in the corresponding genes.
Keywords: Y. enterocolitica , resistance mechanisms , ß-lactams , molecular characterization
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Introduction |
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Most Yersinia enterocolitica strains are known to produce two different chromosomally encoded ß-lactamases, ß-lactamase A (a constitutive broad-spectrum enzyme hydrolysing carbenicillin) and ß-lactamase B (an inducible cephalosporinase).1 The genes encoding both ß-lactamases have been cloned and characterized from different clinical strains of Y. enterocolitica, namely the blaA gene from strain Y56, which only expressed BlaA, and the blaB (ampC) gene from strain IP97, which only expressed BlaB or AmpC.2 Sequence data showed that BlaA belongs to molecular class A,3 while BlaB is an AmpC-type ß-lactamase belonging to molecular class C.4
The Y. enterocolitica ampC gene is transcriptionally regulated by the divergently expressed ampR,4 as occurs in other members of the family Enterobacteriaceae. When Yersinia strains were analysed by Southern hybridization, the two ß-lactamase genes were found uniformly present in their genomes, however, not all Y. enterocolitica strains expressed the two ß-lactamases simultaneously.5,6 In this study, we have analysed the molecular basis of this phenomenon and have discussed possible explanations for this unusual situation.
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Materials and methods |
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Bacterial strains and susceptibility testing
The Y. enterocolitica strains used in this work were Y56, H6, Y60 and H14 of the 4/O:3/VIII group, and IP4119, IP4124, IP22273 and IP22274 of the 4/O:3/IXb group containing active blaA and inactive ampC genes; and strains IP97 (2/O:5b/Xz group) containing inactive blaA but active ampC genes; and P1403 (1A/O:5a/Xo group) containing inactive blaA and ampC genes. Antibiotics were purchased from Sigma (St Louis, MO, USA). The MICs of the ß-lactam antibiotics were determined on Mueller–Hinton agar (Difco Laboratories, Detroit, MI, USA) containing 2-fold serial dilutions of the antibiotics. The inoculum size was 104 cfu/mL.
Cloning of inactive ß-lactamase genes
Chromosomal DNA from strains carrying inactive bla genes was hybridized with probes obtained from the corresponding active genes. DNA fragments containing the genes were cloned into a plasmid vector and recombinant plasmids pSU628 (2.2 kbp EcoRI fragment containing inactive ampC gene from strain Y56, ampC56) and pSU712 (12 kbp EcoRI/HindIII fragment containing inactive blaA gene from strain IP97, blaA97) were obtained.
ß-Lactamase assays
ß-Lactamase activity in crude extracts was determined spectrophotometrically with 100 µM cefalotin as substrate, essentially as described previously.2 The kinetic parameters, Km and Vmax, were determined as recommended7 using five different concentrations of cefalotin (from 20 to 100 µM).
DNA sequencing, PCR amplification and sequence analysis
DNA sequencing was performed with a DNA automatic sequencer (Vistra 725; Amersham, UK). The ampC56 sequence was determined using plasmid pSU628 as template and the appropriate primers.
A 608 bp ampC internal region from several Y. enterocolitica strains was amplified using BlaB-2 (5'-CCGCAATTCTTTAACT; positions 1734–1749; GenBank X63149 [GenBank] ) and BlaB-3 (5'-GGGTCCATATTGGCCTGC; positions 2324–2341; GenBank X63149 [GenBank] ) as primers. PCR were carried out with an initial denaturation step of 5 min at 95°C followed by 30 cycles of 30 s of denaturation at 94°C, 30 s of annealing at 40°C and 30 s of extension at 72°C, followed by a final extension for 10 min at 72°C. The corresponding products were sequenced and compared with the ampC97 sequence in order to determine the presence of mutations.
The sequence of the blaA gene from strain IP97, blaA97, was determined by using pSU712 as template and BlaA-1 (5'-TGACTTTATGCATTATTA; positions 301–318 of the blaA gene sequence from strain Y56, GenBank X57074 [GenBank] ), BlaA-2 (5'-CATGACCGTCTCCGAATT; positions 700–717; GenBank X57074 [GenBank] ) and BlaA-3 (5'-GTAACATCACCCGCACAG; positions 1227–1244; GenBank X57074 [GenBank] ) as primers.
The sequences were analysed using the program BLASTN (version 2.2.3) in the Blast server at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST).
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Results |
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Sequencing of the inactive blaA gene from Y. enterocolitica strain IP97
The sequence of the blaA97 gene was determined and compared with the sequence of the blaA gene from strain Y56 (blaA56, GenBank X57074 [GenBank] ). A deletion of 51 bp was observed, which produced the in-frame loss of 17 amino acids in the ß-lactamase open reading frame (Figure 1). No more changes were observed in the nucleotide sequence of the blaA97 gene. MIC and in vitro activity determinations carried out using crude extracts showed that the BlaA97 enzyme was completely inactive.
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The genome of Y. enterocolitica strain P1403, which also encodes an inactive blaA gene,6 was analysed by PCR and a normal sized amplicon was found instead. The reason for BlaA inactivation in this strain remains unknown.
Cloning and analysis of the ampR-ampC genes from Y. enterocolitica strain Y56
To investigate the lack of AmpC activity in Y. enterocolitica strain Y56, the nucleotide sequence of the ampC-ampR region was determined and compared with the same region from strain IP97. A mutation was found at position 2298 (as reported in GenBank X63149 [GenBank] ). The A to G change at this position resulted in the substitution of a threonine with an alanine at position 251 in AmpC56. A second mutation (G to A) was located at position 2531. However, this mutation did not produce an amino acid change.
ampC and ampR genes from Y56 and IP97 strains were separately cloned to prove that the mutation A751G, and not a mutation elsewhere, was responsible for the lack of AmpC activity in Y56. Then, plasmids combining ampC and ampR regions from both strains were constructed. The MICs of a number of ß-lactam antibiotics for Escherichia coli DH5
carrying the resulting recombinant plasmids were evaluated as indicated in the Materials and methods section. The results are shown in Table 1. Full-level resistance was only observed when the ampC gene from strain IP97 was present independently of the origin of the ampR region (which also contained the promoters for both ampC and ampR genes). These results suggested that both ampR and the intergenic control region were functional in Y. enterocolitica strain Y56 and therefore the lack of AmpC activity in this strain should be due to the changes in the ampC structural gene (ampC56).
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To further demonstrate this point, we constructed two chimeric ampC genes by exchanging the 3' half containing the detected mutations between plasmid pSU628 (carrying ampC56) and pSU641 (carrying the corresponding region from ampC97). The resulting recombinant plasmids were called pSU642 and pSU643, respectively. The MIC of ampicillin for E. coli RR1
M15 carrying the plasmids pSU628 (ampC56) or pSU643 (chimeric gene with 3' half of ampC56) was 8 mg/L. However, the MIC of ampicillin for E. coli RR1M15 carrying pSU641(ampC97) or pSU642 (chimeric gene with 3' half of ampC97) was 128 mg/L. This result indicated that the presence of the A751G mutation was associated with an ampicillin-susceptible phenotype and that this mutation was responsible for the inactivation of the ampC gene in strain Y56.
Identification of mutations in other inactive ampC genes from Y. enterocolitica strains
In order to determine whether the mutation at position 751 in the ampC56 gene was responsible for the lack of cephalosporinase activity in other Y. enterocolitica strains, an ampC internal 608 bp region of several strains showing an inactive AmpC was amplified by PCR using the primers BlaB-2 and BlaB-3 and their nucleotide sequences were determined. The same mutations (G984A and A751G) were found in the closely related strains Y60, H6 and H14. In contrast, the four strains belonging to the 4/O:3/IXb group showed the same 2 bp deletion (CG at position 755–756) that introduced a change of the reading frame leading to misreading of the protein after amino acid 252 and to a premature stop. The strain P1403 presented several nucleotide changes in the same region, although only a mutation in the ampC-coding sequence (A646G) resulted in an amino acid substitution, changing an isoleucine for a valine at position 216 of AmpC ß-lactamase. This change was disregarded as the cause of P1403 AmpC inactivation because valine is present in most of the enterobacteria AmpC ß-lactamases.8
Kinetic properties of the AmpC ß-lactamase from the Y. enterocolitica strains Y56 and IP97
Crude protein extracts from E. coli RR1M15 harbouring plasmids pSU628 (containing the ampC56 gene) and pSU630 (containing the ampC97 gene) were assayed for ß-lactamase activity using cefalotin as substrate. The Km values for the AmpC97 and AmpC56 cephalosporinases were 20.13 and 14.15 µM, respectively, which suggests that the affinity for cefalotin was similar in both enzymes. However, when the Vmax was analysed, a big difference was observed for both AmpC enzymes. Vmax values were 0.97 µmol per min per mg of protein and 0.02 µmol per min per mg of protein, respectively.
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Discussion |
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Members of the genus Yersinia are well-known ß-lactamase producers. Previous biochemical and genetic studies have shown that Y. enterocolitica isolates may produce in some cases two different ß-lactamases, only one or none of them.5,9 This characteristic seems to be common to other yersiniae, which are likely to be involved in human disease.10 However, the genes encoding both enzymes can be found in the chromosome of all the strains examined,6 indicating that some ß-lactamase genes could be either inactivated by mutations or not expressed under regular conditions.
In this study we have identified the causes of inactivation of the ß-lactamase genes blaA and ampC of Y. enterocolitica strains IP97 and Y56, respectively. In the case of blaA from strain IP97 (blaA97), a deletion of 51 bp resulted in a completely inactive ß-lactamase. In ampC56 (ampC from strain Y56), two mutations were found. One of them was silent, while the other, producing the substitution of a threonine with alanine, reduced the cefalotin hydrolysis capacity (Vmax/Km) of AmpC56 to 3% with respect to the wild-type enzyme AmpC97. This mutation might affect the structure of the AmpC56 enzyme producing a practically inactive AmpC ß-lactamase with a Km similar to AmpC97 but a much lower Vmax.
The mutation A751G was also found in three other Y. enterocolitica strains (Y60, H6 and H14), which is consistent with the lack of activity of AmpC in these strains. A different mutation consisting of the deletion of 2 nt (CG at position 755–756) was found in the ampC gene from another four strains. This deletion introduced a change in the reading frame leading to a frameshift after amino acid 252 and to a premature stop. All the ampC mutations studied here could be located on the loop structure between -8 and -9 helices, which has been described as a hot-spot for extended substrate specificity mutations,11 common among class C ß-lactamases.
Accumulation of several ß-lactamases in the same bacterium is usual in clinical isolates and contributes to the broadening of the resistance profile to ß-lactam antibiotics.12 It is widely accepted that this multiplicity constitutes an evolutionary advantage for bacteria that are usually in contact with antibiotics.
Y. enterocolitica strains seem to behave against this rule suggesting that the simultaneous production of a class A and a class C ß-lactamase may encompass, under some circumstances, a decrease in fitness leading to the inactivation of one of the two enzymes.
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None to declare.
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Acknowledgements |
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This work was supported by grants from the Spanish ‘Ministerio de Sanidad y Consumo’ to J. M. G. L. (PI050894/05) and from the ‘Fundación Marqués de Valdecilla-IFIMAV’ to M. V. F. (API04/12).
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References |
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Nukaga M, Haruta S, Tanimoto K, et al. (1995) Molecular evolution of a class C ß-lactamase extending its substrate specificity. J Biol Chem 270:5729–35.
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Fosse T, Giraud-Morin C, Madinier I, et al. (2004) Aeromonas hydrophila with plasmid-borne class A extended-spectrum ß-lactamase TEM-24 and three chromosomal class B, C, and D ß-lactamases, isolated from a patient with necrotizing fasciitis. Antimicrob Agents Chemother 48:2342–3.
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