DEVELOPMENT OF PCR ASSAYS TO DETECT AMPICILLIN RESISTANCE GENES IN CEREBROSPINAL-FLUID SAMPLES CONTAINING HAEMOPHILUS-INFLUENZAE

We developed PCR primers specific for the bla(TEM) and bla(ROB) ampicillin resistance genes. The specificity of the primers was confirmed by testing a series of Escherichia coli isolates containing a variety of ampicillin resistance genes and a series of ampicillin-resistant and ampicillin-susceptible Haemophilus influenzae isolates. There was a perfect correlation between ampicillin MICs, the presence of beta-lactamase (as determined by the nitrocefin test), and the results with the bla(TEM) and bla(ROB) primers. Isolates of H. influenzae and Streptococcus pneumoniae obtained from 25 frozen cerebrospinal fluid (CSF) specimens were also tested. Four of 14 H. influenzae isolates were positive with the bla(TEM) primers; none were positive with the bla(ROB) primers. Ampicillin MICs were determined for the H. influenzae isolates, and penicillin MICs were determined for the S. pneumoniae isolates. Only the four PCR-positive H. influenzae isolates had elevated MICs of ampicillin and were beta-lactamase positive. None of the H. influenzae isolates contained the bla(ROB) gene, and none of the S. pneumoniae isolates produced positive reactions with either primer set. We then used universal primers directed to conserved regions of rRNA and a Haemophilus detection probe to identify which of the 25 frozen samples of CSF contained H. influenzae. Fourteen of the 25 CSF specimens,vere positive for H. influenzae, which correlated with the number of organisms obtained by culture of the CSF samples. Four of the CSF samples were positive with the bla(TEM) primer set, and these correlated with the four H. influenzae isolates that were positive when tested directly by PCR. The bla(TEM) assay required the use of native Tag polymerase because Amplitaq preparations were contaminated with vector DNA that contained the bla(TEM-1) gene.