| Summary: | The gene encoding TEM-E2 (<I>bla</I>T-E2), an ESBL identified in an isolate of <I>Klebsiella oxytoca</I> responsible for an outbreak of infection in a neonatal unit in Liverpool in 1982, was sequenced. As cloning from PCR-amplified DNA fragments proved difficult, a new technique for direct DNA-sequencing of PCR-amplified DNA fragments was developed. From the sequences of <I>bla</I>T-E2<SUB>pUK721</SUB> generated by this method, a new nucleotide was identified at DNA residue number 691, corresponding to adenosine. Biochemical experiments and PCR studies showed that the gene for TEM-E2 was present in addition to TEM-1. No additional bands were visible in the same regions of TEM-1 control sequences produced by the same method. In conclusion: Conservative mutations or mutations remote from the active site do not affect biological parameters in a measurable manner. Evolutionary implications of the single mutations may be relatively minor and may not be detected during the normal course of events. Silent mutations, or minimally important mutations, occur frequently with little functional consequence but may prepare bacteria to be ready for a more drastic change when challenged with a 3GC. Acquisition of a single innocuous mutation facilitates the occurrence of a more dangerous double mutation. The results suggest that clavulanic acid, in combination with amoxycillin, promotes back mutations from extended-spectrum β-lactamases to TEM-1 by a mechanism of gene duplication facilitated by intermolecular transposition. I have suggested the hypothesis that the evolution of the parent β-lactamase to broad-spectrum enzyme can consist of both forward and backward mutations at well defined locations. This hypothesis is supported by my results which show that amoxycillin in combination with clavulanic acid selects back mutations to TEM-1 and other extended-spectrum β-lactamases. The back mutations are facilitated by gene duplication. The host organism will also contain a heterogeneous population of a clinical plasmid. The point-mutations occur in the TEM-gene during duplication of the gene. In a non-selective environment one of the duplicate genes is usually deleted.
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