We report a case of necrotizing fasciitis with probable in vivo transfer of a TEM-24 plasmid-borne extended-spectrum β-lactamase (ESBL) gene from Enterobacter aerogenes to Aeromonas hydrophila. The patient was an 87-year-old female with a leg lesion following a trauma. She had a history of rheumatoid polyarthritis treated by 10 mg of prednisone per day, refractory anemia, and chronic venous insufficiency of the lower limbs. Within 5 days, the infection grew worse and the initial amoxicillin-clavulanic acid antibiotic therapy was replaced with ceftriaxone-metronidazole (1 to 1.5 g daily). Surgical debridement revealed extensive necrosis, and 3 days later, the lesion evolved toward typical necrotizing fasciitis (1, 6), leading to a second surgical intervention for above-knee amputation followed by complete healing.
Routine bacteriological procedures revealed (i) Escherichia coli NI-202 susceptible to most β-lactam compounds, (ii) E. aerogenes NI-203 resistant to all β-lactam antibiotics except imipenem, (iii) A. hydrophila NI-204 resistant to ceftazidime, and (iv) A. hydrophila NI-205 susceptible to ceftazidime (Table 1). Pulsed-field electrophoresis confirmed that A. hydrophila NI-204 and NI-205 derived from a single clone. For β-lactamase analysis, the E. aerogenes isolate was grown in brain heart infusion broth with and without cefoxitin or ceftazidime induction (10 μg/ml) at 37°C before analytical isoelectric focusing with crude sonic cell extracts on polyacrylamide gels (2, 4). Two bands of β-lactamase activity were detected with iodine gel with cefazolin (500 μg/ml) as the substrate, which was suggestive of the production of an inducible cephalosporinase (pI 8.8) and an ESBL (pI 6.5). Aeromonas isolates were grown at 30°C with cefoxitin (10 μg/ml), imipenem (1 μg/ml), or tobramycin (1 μg/ml) induction (5). Analytical isoelectric focusing with penicillin and cefazolin as substrates revealed three bands (pI 7, 7.8, and 8.2) probably corresponding to previously described cephalosporinase-, imipenemase-, and oxacillinase-type inducible β-lactamases (5, 11, 12). A. hydrophila NI-204 produced an additional enzyme similar to E. aerogenes NI-203 ESBL (pI 6.5).
TABLE 1.
β-Lactamases, plasmid content, and MICs (μg/ml) of the clinical isolates and transconjugant strains
Parameter or drug(s) | E. aerogenes NI-203 | A. hydrophila NI-204 | A. hydrophila NI-205 | A. hydrophila NI-206 | E. coli C1a | E. coli C1a NI-207 |
---|---|---|---|---|---|---|
pI | 6.5, 8.8 | 6.5, 7, 7.8, 8.2 | 7, 7.8, 8.2 | 6.5, 7, 7.8, 8.2 | None | 6.5 |
Plasmid size (kb) | 180 | 180 | 180 | 180 | ||
PCR TEM | + | + | − | − | − | + |
Amoxicillin | >256 | >256 | >256 | >256 | 2 | >256 |
Amoxicillin + clavulanic acid | 8 | 8 | 8 | 16 | 2 | 2 |
Ticarcillin | >256 | >256 | >256 | >256 | 8 | >256 |
Ticarcillin + clavulanic acid | 64 | 32 | 64 | 16 | 8 | 8 |
Piperacillin | 256 | 4 | 2 | 4 | 2 | 64 |
Piperacillin + tazobactam | 16 | 1 | 1 | 1 | 2 | 2 |
Cefoxitin | >256 | 16 | 16 | 2 | 2 | 2 |
Cefepime | 1 | 0.25 | 0.06 | 0.25 | ≤0.03 | 1 |
Cefepime + clavulanic acid | 0.125 | ≤0.03 | ≤0.03 | ≤0.03 | ≤0.03 | ≤0.03 |
Cefuroxime | >256 | 2 | 1 | 2 | 1 | 1 |
Cefpirome | 2 | 0.5 | 0.03 | 0.5 | 0.03 | 1 |
Cefotaxime | >32 | 0.5 | 0.06 | 0.5 | 0.06 | 1 |
Ceftazidime | >256 | 32 | 0.125 | 32 | 0.125 | 256 |
Ceftazidime + clavulanic acid | >4 | 0.06 | 0.06 | 0.06 | 0.25 | 0.25 |
Imipenem | 0.5 | 4 | 4 | 8 | 0.5 | 0.5 |
Amikacin | 8 | 8 | 2 | 8 | 0.25 | 16 |
Tobramycin | 8 | 16 | 2 | 16 | 0.25 | 16 |
Sulfamethoxazole-trimethoprim | >32 | >32 | 0.25 | >32 | 0.5 | >32 |
Ciprofloxacin | >32 | ≤0.03 | ≤0.03 | ≤0.03 | 0.125 | 0.125 |
Taking into account resistance to ceftazidime, pI determination, and local epidemiology, the ESBL was presumed to be the plasmid-mediated TEM-24 βlactamase (2-4, 7). The plasmid was transferred from E. aerogenes NI-203 to A. hydrophila NI-205 and to E. coli C1a at a high frequency (10−4). Recipient strains (NI-206 and NI-207, Table 1) presented the same acquired resistance pattern. After plasmid extraction and gel electrophoresis, both wild-type strains (E. aerogenes NI-203, A. hydrophila NI-204) and recipient strains (A. hydrophila NI-206, E. coli C1a NI-207) showed a common 180-kb band, as previously characterized with Enterobacteriaceae, Pseudomonas aeruginosa, and recently A. caviae (4, 7-9). The capacity of Aeromonas salmonicida to maintain either or both of the Pseudomonas and Enterobacteriaceae R factors has already been observed (10). PCR amplification with TEM family-specific primers was applied to E. aerogenes NI-203 and A. hydrophila NI-204 and showed a deduced protein sequence with 100% identity to that of TEM-24 (3, 7).
This report demonstrates probable in vivo transfer of ESBL TEM-24 from E. aerogenes to the genus Aeromonas. It was observed in a wild-type strain of A. hydrophila simultaneously producing the class A, B, C, and D β-lactamases.
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