Identification by DNA sequence analysis of a new plasmid-encoded trimethoprim resistance gene in fecal Escherichia coli isolates from children in day-care centers

In our ongoing studies of trimethoprim resistance (Tmp(r)) in day-care centers (DCC), we have shown a high rate of fecal colonization with Tmp(r) Escherichia coli and, using total plasmid content analysis, have shown that this is due to a diversity of strains. In the present study, we analyzed 367 highly Tmp(r) (MIC, ≥2,000 μg/ml) isolates of E. coli from 72 children over a 5-month period and found at least 83 distinct plasmid patterns, indicating that at least 83 strains were involved. Several strains were particularly common in a given DCC, including one found in 61% of children with Tmp(r) E. coli; these common strains usually persisted within a DCC for several months. Colony lysates were hybridized with gene probes for dihydrofolate reductases (DHFR) types I, II, III, V, and VII; 21% hybridized under stringent conditions, and all of these were with type I (17%) or type V (4%) probes. Tmp(r) was cloned from a probe-negative Tmp(r) transconjugant, and an intragenic probe was prepared from this clone. Approximately 21% of the Tmp(r) E. coli strains (76 isolates) in the DCC were found to have this new gene, 74 of which were in one DCC. The DNA sequence of this gene was determined, and the predicted amino acid sequence was shown to have between 32% and 39% identity with the amino acid sequences for types I, III, V, VI, and VII and the partial sequence of type IV and ~26% identity with types IX and X DHFR. This confirms the uniqueness of this gene, which has tentatively been named dhfrxii, and its translation product, DHFR type XII.