Sequences of small blaTEM-encoding plasmids in Haemophilus influenzae and description of variants falsely negative for blaTEM by PCR

Objectives: To characterize an unidentified β-lactamase and associated genetic background in a bla_TEM and bla_ROB PCR-negative Haemophilus influenzae isolate, and characterize small bla_TEM-encoding plasmids in a collection of H. influenzae.

Methods: The unidentified β-lactamase gene was identified by cloning and sequencing the encoding plasmid. Strains with small bla_TEM plasmids were identified using negative PCR for integrative conjugative elements, but positive bla_TEM PCR; plasmids from selected isolates were sequenced. PCR for rep and divergent bla_TEM were evaluated for detecting small plasmids on selected H. influenzae isolates.

Results: Small plasmids (4.8-5.5 kb) encoding bla_TEM appear to be associated with remnants of Tn2 on a conserved plasmid core. The unidentified β-lactamase was actually a TEM-1, with negative bla_TEM PCR associated with a previously unrecognized deletion of bp 1-27 of bla(TEM) (Sutcliffe numbering) associated with a larger deletion within Tn2. This deletion was found in other isolates and may be more common than previously thought. PCR for the conserved rep gene appears useful for screening for small bla_TEM-encoding plasmids or associated cryptic plasmids in H. influenzae.

Conclusions: Small bla_TEM-encoding plasmids in H. influenzae appear relatively conserved, but require further study to confirm this. PCR associated with the rep gene may be useful for studying these small plasmids. A deletion in part of bla_TEM in some strains may interfere with some PCRs; therefore, care should be taken with primer selection or design and, preferably, regions within the open reading frame should be targeted.