In vivo selection of a complex mutant TEM (CMT) from an inhibitor-resistant TEM (IRT) during ceftazidime therapy

A relapse from Escherichia coli bloodstream infection was observed in a patient with acute leukaemia treated with ceftazidime for 7 days for febrile neutropenia. Whereas the original E. coli isolate was resistant to β-lactam/β-lactamase inhibitor combinations (EC1), the relapse E. coli isolate showed a similar phenotype but with resistance extended to ceftazidime (EC2). We investigated the molecular mechanisms of β-lactam resistance and sought if EC2 could have been selected in vivo from EC1.

EC1 and EC2 isolates were compared for antibiotic MICs, plasmid content, genotyping, β-lactamase genes and their environment. Both isolates were conjugated with E. coli JW4111ΔampC and MICs determined for transconjugants. In addition, ceftazidime-resistant mutants were selected in vitro from EC1.

EC1 and EC2 showed identical patterns for genotyping and resistance plasmids. PCR sequencing of blaTEM in EC1 showed the mutations M69L and N276D corresponding to TEM-35, also called inhibitor-resistant TEM (IRT)-4. In EC2, the TEM allele showed an additional mutation, R164S, known to confer resistance to ceftazidime. The combination of these three mutations was previously reported in TEM-158, described as the complex mutant TEM (CMT)-9, associated with resistance to β-lactamase inhibitors and third-generation cephalosporins. In vitro selection of ceftazidime-resistant mutants from EC1 yielded six different CMT alleles, including TEM-158 containing the R164S mutation.

This first known report of in vivo selection of CMT from IRT, reproduced in vitro, shows how the evolution of β-lactamase enzymes is easily driven by antibiotic pressure, even during a short antibiotic therapy.