Molecular characterization of TEM-type β-lactamases identified in cold-seep sediments of edison seamount (South of Lihir Island, Papua New Guinea)

To determine the prevalence and genotypes of β-lactamases among lones of a metagenomic library from the cold-seep sediments of Edison seamount (10,000 years old), we performed pulse-field gel electrophoresis, antibiotic susceptibility testing, pI determination, and DNA sequencing analysis. Among the 8,823 clones of the library, thirty clones produced ß-lactamases and had high levels of genetic diversity. Consistent with minimum inhibitory concentration patterns, we found that five (16.7%) of thirty clones produced an extended-spectrum β-lactamase. 837- and 259-bp fragments specific to bla_TEM genes were amplified, as determined by banding patterns of PCR amplification with designed primers. TEM-1 1 was the most prevalent β-lactamase and conferred resistance to ampicillin, piperacillin, and cephalothin. TEM-116 had a spectrum that was extended to ceftazidime, cefotaxime, and aztreonam. The resistance levels conferred by the pre-antibiotic era alleles of TEM-type β-lactamases were essentially the same as the resistance levels conferred by the TEM-type alleles which had been isolated from clinically resistant strains of bacteria of the antibiotic era. Our first report on TEM-type β-lactamases of the pre-antibiotic era indicates that TEM-type β-lactamases paint a picture in which most of the diversity of the enzymes may not be the result of recent evolution, but that of ancient evolution.