Abstract
The sequences of the blaTEM genes encoding TEM-92 in Proteus mirabilis and Providencia stuartii isolates were determined and were found to be identical. Except for positions 218 (Lys-6) and 512 (Lys-104), the nucleotide sequence of blaTEM-92 was identical to that of blaTEM-20, including the sequence of the promoter region harboring a 135-bp deletion combined with a G-162→T substitution. The deduced amino acid sequence of TEM-92 differed from that of TEM-52 by the presence of a substitution (Gln-6→Lys) in the peptide signal.
The extended-spectrum beta-lactamases (ESBLs) observed in Proteus mirabilis and rarely in Providencia stuartii are often difficult to detect. Their detection requires a modified synergy test (16) because they are usually produced at low levels (5).
We report here on two strains, P. mirabilis CF 529 and P. stuartii 1606 (Table 1), isolated in 1998 from the urinary tracts of two patients hospitalized at Clermont-Ferrand Hospital and Pitié-Salpêtrière Hospital in Paris, France, respectively. These isolates, in particular, P. mirabilis CF 529, were noticed because of their high level of resistance to cefotaxime and the results of the synergy test, which was unequivocally positive with oxyiminocephalosporins and clavulanic acid.
TABLE 1.
ESBL-producing clinical strains used in this study
| Strain | Reference | Beta-lactamase produced (location, yr of isolation) | Isoelectric point |
|---|---|---|---|
| P. mirabilis CF 529 | This study | TEM-92 (Clermont-Ferrand, 1998) | 6.0 |
| P. stuartii 1606 | This study | TEM-92 (Paris, 1998) | 6.0 |
| P. mirabilis TrCF529 | This study | TEM-92 (transconjugant) | 6.0 |
| P. mirabilis CF 39 | 3 | TEM-3 (Clermont-Ferrand, 1996) | 6.3 |
| P. mirabilis CF 669 | 3 | TEM-66 (Clermont-Ferrand, 1997) | 6.0 |
| K. pneumoniae NEM 865 | 11 | TEM-52 (Paris, 1996) | 6.0 |
MICs were determined by a dilution method on Mueller-Hinton agar with an inoculum of 104 CFU per spot (6). Antibiotics were provided as powders by SmithKline Beecham Pharmaceuticals (amoxicillin, ticarcillin, and clavulanate), Eli Lilly, Paris, France (cephalothin, moxalactam), Roussel-Uclaf (cefotaxime, cefpirome), Glaxo-Wellcome Research and Development (ceftazidime), Bristol-Myers Squibb (aztreonam, cefepime), and Merck Sharp & Dohme (cefoxitin).
Table 2 lists the MICs of aztreonam, cefoxitin, cefotaxime alone and combined with clavulanate at a fixed concentration of 2 μg/ml, ceftazidime, cefepime, cefpirome, and moxalactam for the two strains producing TEM-92, P. mirabilis CF 529 and P. stuartii 1606. They were compared with those for P. mirabilis CF 39 and P. mirabilis CF 669, two strains of the same species that produce TEM-3 and TEM-66, respectively (3). These three ESBLs, TEM-92, TEM-3, and TEM-66, have the same mutations, Glu-104→Lys and Gly-238→Ser, implicated in the extension of the spectrum of activity.
TABLE 2.
MICs for the clinical isolates of ESBL-producing P. mirabilis and P. stuartii
| Strain (beta-lactamase) | MIC (μg/ml)a
|
|||||||
|---|---|---|---|---|---|---|---|---|
| ATM | FOX | CTX | CTX + CLA | CAZ | FEP | CPO | MOX | |
| P. mirabilis CF529 (TEM-92) | 0.5 | 8 | 64 | 0.12 | 8 | 32 | 16 | 0.25 |
| P. stuartii 1606 (TEM-92) | 2 | 2 | 32 | ≤0.06 | 8 | 2 | 2 | 0.25 |
| P. mirabilis CF 39 (TEM-3) | 0.06 | 4 | 2 | 0.06 | 0.5 | 0.25 | 2 | 0.25 |
| P. mirabilis CF 669 (TEM-66) | 0.25 | 8 | 8 | 0.06 | 8 | 0.12 | 16 | 0.25 |
ATM, aztreonam; FOX, cefoxitin; CTX, cefotaxime; CLA, clavulanic acid (2 μg/ml); CAZ, ceftazidime, FEP, cefepime; CPO, cefpirome; MOX, moxalactam.
P. mirabilis CF 529 and P. stuartii 1606 differed from P. mirabilis strains CF 39 (TEM-3) and CF 669 (TEM-66) in that the first two strains had higher levels of resistance to cefotaxime (MICs for CF 529 and 1606, 64 and 32 μg/ml, respectively; MICs for CF 39 and CF 669, 2 and 8 μg/ml, respectively). The ceftazidime MIC was 8 μg/ml for all strains except P. mirabilis CF 39 (TEM-3), for which it was 0.5 μg/ml.
Aztreonam MICs (≤2 μg/ml) remained low. The four strains were susceptible to moxalactam (MICs, ≤0.25 μg/ml) and cefoxitin (MICs, ≤8 μg/ml), unlike Klebsiella pneumoniae NEM 865 producing TEM-52 (14). Clavulanate restored the impaired activity of cefotaxime. Analytical isoelectric focusing was performed with crude lysates from polyacrylamide gels containing ampholines with a pH range of 3.5 to 10.0, as described previously (15). Both of the clinical strains (P. mirabilis CF 529 and P. stuartii 1606) produced a beta-lactamase with a pI of 6.0.
Several transfer experiments were tried with mutants of Escherichia coli HB 101, E. coli C600, and P. mirabilis ATCC 29906 as recipient strains. Only one transconjugant strain was obtained by mating P. mirabilis CF 529 with rifampin-resistant P. mirabilis ATCC 29906. The phenotype of resistance to aminoglycosides (kanamycin, tobramycin, and gentamicin) observed by the diffusion method was cotransferred with the ESBL phenotype.
Plasmids from clinical isolates and the transconjugant were extracted by the method of Kado and Liu and electrophoresed at 250 V for 4 h in a 0.7% agarose gel. They were blotted onto Nytran filters.
Hybridization with a TEM probe obtained by PCR with primers TEM-A (5′-TAAAATTCTTGAAGACG-3′) and TEM-B (5′-TTACCAATGCTTAATCA-3′) (3) and labeled by random priming (DNP-DNA labeling kit; Appligen Oncor, Illkirch, France) showed that the TEM gene resided on a 50-kb plasmid of P. mirabilis CF 529 and P. stuartii 1606 (data not shown). In K. pneumoniae NEM 865 (TEM-52), β-lactam resistance was transferred alone, without aminoglycoside resistance genes, and was located on a 13.5-kb plasmid. Great variability in plasmid size in TEM-52-producing E. coli and K. pneumoniae strains (between 71 and 100 kb) was also observed in Korea (12). PCR amplification and DNA sequencing of the promoter and coding regions of the blaTEM-92 gene were performed as described previously (3).
The nucleotide sequences of the two blaTEM genes from P. mirabilis CF 529 and P. stuartii 1606 were identical. Analysis of the deduced protein sequence compared to that of blaTEM-1 showed four amino acid substitutions, Gln-6→Lys, Glu-104→Lys, Met-182→Thr, and Gly-238→Ser. This protein sequence is identical to that of TEM-52 reported previously (12, 14) except for the substitution Gln-6→Lys in the signal peptide. We suggest that the enzyme be designated TEM-92.
In Table 3, which shows the positions known to allow discrimination of blaTEM genes (10), the sequence of blaTEM-92 is compared with those of blaTEM-52 genes reported previously (12, 14) and with that of blaTEM-20 (2). blaTEM-92 and blaTEM-20 have the same promoter with a 135-bp deletion between nucleotides 22 and 158 combined with the mutation G-162→T. This combination of the deletion and the mutation resulted in a promoter sequence that contained TTGAA for the −35 region and TACAAT for the −10 region and that is thereby of closer similarity to the consensus promoter sequence, which conferred a strong promoter (2). No deletion was observed in the promoters of blaTEM-52 genes reported previously, in which strong promoters, P4 (G-162→T) for K. pneumoniae NEM 865 (14) and Pa + Pb (C-32→T) for K. pneumoniae KMK 107 (BLAST program, National Center for Biotechnology Information, accession number AF 027 199 [http://www.ncbi.nlm.nih.gov/BLAST/]), were observed.
TABLE 3.
Nucleotide and amino acid substitutions of blaTEM-92 and related blaTEM genes
| Region and nucleotide no.a (amino acid no.b) | Nucleotide (amino acid) in the following genes
|
||||||
|---|---|---|---|---|---|---|---|
| blaTEM-1ad | blaTEM-52ae | blaTEM-1bd | blaTEM-52bf | blaTEM-2d | blaTEM-20g | blaTEM-92 (this study) | |
| Promoter | |||||||
| 32 | C | C | C | T | T | Deleted | Deleted |
| 147 | T | T | T | T | T | Deleted | Deleted |
| 162 | G | T | G | G | G | T | T |
| 175 | A | A | G | G | A | A | A |
| Gene | |||||||
| 218 (6) | C (Gln) | C | C | C | C | C | A (Lys) |
| 226c | C | C | T | T | C | C | C |
| 317 (39) | C (Gln) | C | C | C | A (Lys) | C | C |
| 346c | A | A | A | A | G | G | G |
| 436c | C | C | T | T | T | T | T |
| 512 (104) | G (Glu-104) | A (Lys) | G | A (Lys) | G | G | A (Lys) |
| 604c | G | G | T | T | G | G | G |
| 682c | T | T | T | T | C | C | C |
| 747 (182) | T (Met) | C (Thr) | T | C (Thr) | T | C (Thr) | C (Thr) |
| 914 (238) | G (Gly) | A (Ser) | G | A (Ser) | G | A (Ser) | A (Ser) |
| 925c | G | G | G | G | A | A | A |
If we consider the silent mutations at positions 226, 346, 436, 604, 682, and 925 known to allow the discrimination of blaTEM genes, blaTEM-92 is related to blaTEM-2 (10). In comparison, blaTEM-52 from K. pneumoniae NEM 865 was identical to blaTEM-1a, and blaTEM-52 from K. pneumoniae KMK 107 was identical to blaTEM-1b. Compared to the blaTEM-15 gene (10), we could designate the gene from K. pneumoniae NEM 865 blaTEM-52a and the gene from K. pneumoniae KMK 107 blaTEM-52b. If we consider the sequences of the promoter and coding regions, blaTEM-92 was identical to blaTEM-20 (2) except at two positions, 218 and 512 (Glu-6→Lys and Glu-104→Lys, respectively), and both genes belong to the blaTEM-2-like group (4).
Among ESBLs, TEM-52 was hitherto not frequent except in Korea, where it was observed in an epidemic (12). It was first reported in a K. pneumoniae strain isolated from a girl originating from Athens, Greece (14), and a strain was observed in France (8). These TEM-52 and TEM-92 enzymes, which harbor the same critical substitutions involved in the extension of the beta-lactamase spectrum at positions 104, 182, and 238, differed by their geographical locations, the species implicated, the sizes of the plasmids carrying the blaTEM gene, and their nucleotide sequences. The occurrence of these enzymes could be due to a convergent evolution from different blaTEM genes (11).
A wide variety of TEM-type ESBLs were observed in P. mirabilis. Some of them (TEM-8, TEM-10, TEM-24, TEM-26 [3], and TEM-87) confer a ceftazidimase resistance phenotype, whereas others (TEM-3, TEM-21, TEM-66 [3] and TEM-72 [13]) confer a cefotaximase resistance phenotype. The latest, TEM-87, has the same mutation (Gln-6→Lys) in the peptide signal as TEM-92. This diversity is perhaps related to the variety of the ecological niches of this species, which is rarely implicated in nosocomial infections (7). In P. stuartii, unlike in P. mirabilis, ESBLs were rarely reported (TEM-24 in our hospital [unpublished data] and TEM-60 [9]). That could be due to the level of expression in this species. A lower level of expression of TEM-92 was observed in P. stuartii 1606 than in P. mirabilis CF 529. For both species we could suspect the existence of a factor that leads to weak expression of β-lactam resistance, despite the presence of a strong promoter (3).
Acknowledgments
We thank Rolande Perroux, Marlène Jan, and Dominique Rubio for technical assistance. We are grateful to C. Poyart, who kindly provided K. pneumoniae NEM 865.
This work was supported in part by a grant from the Direction de la Recherche et des Etudes Doctorales, Ministère de l'Education Nationale de la Recherche et de la Technologie, Paris, France.
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