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Objectives This study was conducted to examine the molecular mechanisms responsible for the evolution of TEM-type extended-spectrum β-lactamases (ESBLs) following selective pressure from four third-generation cephalosporins, namely ceftazidime, cefotaxime, ceftriaxone and ceftibuten. In addition, selective enrichment for ESBL detection in environmental samples was investigated. Methods Using experimental evolution, resistant variants were isolated and mutations in TEM-1 were examined by DNA sequencing. Resistance levels and the development of cross-resistance were determined for ESBL-producing isolates by Etest and disk diffusion assay. Selective plating with or without prior growth in selective broth was used to examine the approach of selective enrichment for ESBL detection. Results The third-generation cephalosporins ceftazidime, cefotaxime and ceftriaxone selected for ESBLs, whereas ceftibuten did not. All ESBL variants additionally remained susceptible to ceftibuten. DNA sequencing of the TEM-1 coding sequence of mutants revealed mutations not previously isolated through selection. This indicates that the potential for ESBL evolution is much broader than can be inferred from sequence analysis of clinical samples alone. The results also indicate that selective enrichment for enhanced detection of ESBL-producers may give unreliable results owing to the selection of spontaneous mutations in narrow-spectrum β-lactamases resulting in TEM-type ESBL-producers. Conclusion These results help explain the molecular changes responsible for evolution of TEM-type ESBLs and meanwhile question the appropriate use of selective enrichment for detection of ESBLs in environmental samples.

The primary objective of this study was to determine the minimum number of individual fecal samples to pool together in order to obtain a representative sample for herd level quantification of antimicrobial resistance (AMR) genes in a Danish pig herd, using a novel high-throughput qPCR assay. The secondary objective was to assess the agreement between different methods of sample pooling. Quantification of AMR was achieved using a high-throughput qPCR method to quantify the levels of seven AMR genes (ermB, ermF, sulI, sulII, tet(M), tet(O) and tet(W)). A large variation in the levels of AMR genes was found between individual samples. As the number of samples in a pool increased, a decrease in sample variation was observed. It was concluded that the optimal pooling size is five samples, as an almost steady state in the variation was observed when pooling this number of samples. Good agreement between different pooling methods was found and the least time-consuming method of pooling, by transferring feces from each individual sample to a tube using a 10μl inoculation loop and adding 3.5ml of PBS, approximating a 10% solution, can therefore be used in future studies.