Ceftazidime resistance evolution in ESBLs belonging to CTX-M-1 cluster (CTX-M-1, CTX-M-3, CTX-M-10) in a hypermutator background

Abstract number: 1732_131

Novais A., Cantón R., Moreira R., Coque T.M., Baquero F., Galán J.C.

Objectives: Resistance to ceftazidime (CAZ) has mostly emerged among enzymes of CTX-M-1 lineage. The aim of this study was to understand evolution of CAZ resistance among CAZ susceptible ESBL belonging to this cluster by step-wise in vitro selection experiments.

Methods: blaCTX-M-1, blaCTX-M-3 and blaCTX-M-10 genes were cloned into pBGS18 plasmid vector (KanR) using EcoRI and PstI restriction enzymes. Recombinant plasmids were further transformed into E. coli strain MI1443 (del-ampC, plasmid free) and its hypermutable isogenic strain GB20 (mutS::Tn10 MI1443). GB20 transformants corresponding to each ESBL were submitted to daily serial passages with increased concentrations of CAZ (0.5–256 fold respect to MIC). Plasmids containing mutants obtained at distinct concentrations were transformed in MI1443 cells, selecting with CAZ (at 4× their corresponding MIC). MICs to CAZ, cefotaxime (CTX), cefuroxime (CXM), cefepime (FEP), and amoxicillin-clavulanate (AMX) were determined in MI1443 carrying the different evolved and non-evolved recombinant plasmids. blaCTX-M genes from strains containing recombinant plasmids displaying reduced susceptibility to CAZ were sequenced.

Results: A high frequency of CAZ mutants was detected (73%; 8/11 transformants tested) with significant increments in CAZ resistance. Interestingly, and with exception of two variants and CXM, a concomitant loss of resistance to all other antibiotics tested (antagonistic pleiotropy) was observed. All mutants contained new blaCTX-M variants except blaCTX-M-52, which evolved from blaCTX-M-3. The most frequent mutations found were D240G and P167S/T, the later conferring higher resistance to CAZ than the former. However, D240G change determined a lower decrease to FEP and CXM than P167S/T. Additionally, two variants were recovered from the evolved blaCTX-M-3 gene: P167S and P167S+A77V, found at 16 and 128 mg/L of CAZ respectively. The double mutant yielded higher resistance level to CAZ, CTX and to a lesser extent to FEP than P167S variant. The A77V change is one of the three polymorphisms between CTX-M-3 and CTX-M-1, whose mutants obtained revealed the highest resistance levels to CAZ and CTX in our study.

Conclusions: This work demonstrates an antagonistic relationship between the susceptibility to CAZ and to other b-lactam antibiotics tested. A77V could be a secondary mutation site affecting CAZ or could be involved in re-equilibrium of CTXR/CAZR co-resistance.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Location: ICC, Munich, Germany
Presentation type:
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