Sequences of small blaTEM-encoding plasmids in Haemophilus influenzae and description of variants falsely negative for blaTEM by PCR.
OBJECTIVE
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.
School of Human Life Sciences, University of Tasmania, Launceston, Tasmania, Australia. stephen.tristram@utas.edu.au
Stephen G
SG
Tristram
Lauren R
LR
Franks
Gareth L
GL
Harvey
D003001:Cloning, Molecular; D004265:DNA Helicases / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D006193:Haemophilus influenzae / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D015534:Trans-Activators / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics*
D017931:DNA Primers; D004269:DNA, Bacterial; D015534:Trans-Activators; C045264:replication initiator protein; D001618:beta-Lactamases; D004265:DNA Helicases