In-vitro activities of five plant essential oils against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium.

Sir, An extended-spectrum, TEM-type -lactamase (ESBL) markedly more active against ceftazidime than other lactams was studied because its kinetic parameters differed from those previously described for ESBLs. None the less, analysis of the nucleotide sequence confirmed the TEM-8 gene. As a first step in attempting to explain this discrepancy, we determined the kinetic parameters of the reference strain synthesizing the TEM-8 ESBL, i.e. the Escherichia coli K-12 C600 transconjugant, CF704, according to a method described previously. When the results were compared with those for the E. coli JM/109 transformant, Tf1 (Table I), they were found to agree. Vmax of the TEM-8 ESBL for ceftazidime (relative to that of benzylpenicillin, which was taken as 100) is therefore 1460 94 (95% confidence interval), rather than 261, the value published earlier; the values for the other kinetic parameters (Table I) were in accord with those reported previously. We then checked the purities of the substrates used in the present study by H-400 MHz NMR and found them (in particular, ceftazidime) to be 95% pure. Unfortunately, none of the ceftazidime used in the original investigation was available. However, we suspect that the purity of this compound may have been low and that this was the explanation for the erroneous results reported initially. The original TEM-8-producing isolate and its transconjugant, CF704, exhibited a greater degree of resistance to ceftazidime than to cefotaxime; the enzyme was therefore designated CAZ-2. The isolate and the transformant, Tf1, which have been studied here exhibited the same resistance phenotype (MICs of cefotaxime and ceftazidime, 64 mg/L and 256 mg/L, respectively). Thus, the results for Vmax are now consistent with the observed MICs. In Table II, the main classification of extendedspectrum, TEM-type -lactamases, based on amino acid substitutions, is related to both the resistance phenotype (determined from the MICs) and to the Payne categorization scheme. When the different amino acid substitutions are examined, it is possible to correlate them with various levels of -lactam resistance, thereby explaining some of the original names of the ESBLs, e.g. CAZ-1 indicating a greater resistance to ceftazidime than to cefotaxime and CTX-1 indicating the reverse. Accordingly, a single mutation at position 164 [serine (Ser) in place of arginine (Arg)] (numbered according to Ambler et al.) leads to a significant level of resistance to ceftazidime (CAZa phenotype), as is the case with TEM-7 and TEM-12. High-level resistance to ceftazidime, cefotaxime and aztreonam (CAZb phenotype) also correlates with an amino acid substitution at position 164 (Ser in place of Arg), but with additional substitutions at positions 104 and/or 237 and/or 240. When the substitution is at position 238 [Ser in place of glycine (Gly)], the resistance phenotype is usually CTX, as for TEM-3. The TEM-8 -lactamase was classified in Payne group 3, which embraces the ESBLs that preferentially hydrolyse cefotaxime. However, according to our results, the TEM-8 -lactamase should be reassigned to group 2 because of its high activity against ceftazidime. Furthermore, TEM-8 has a serine residue at position 238 and a ceftazidimase phenotype, an association not previously recognized. This paradox is presumably attributable to the presence of serine residues at both positions 164 and 238, a combination that is unique among TEM-type ESBLs. We therefore propose the phenotype CAZc for the TEM-8 -lactamase.