Evaluation of multiplex tandem PCR (MT-PCR) assays for the detection of bacterial resistance genes among Enterobacteriaceae in clinical urines

Background: Increasing resistance drives empirical use of less potent and previously reserved antibiotics, including for urinary tract infections (UTIs). Molecular profiling, without culture, might better guide early therapy. 

Objectives: To explore the potential of AusDiagnostics multiplex tandem (MT) PCR UTI assays. 

Methods: Two MT-PCR assays were developed successively, seeking 8 or 16 resistance genes. Amplification was tracked in real time, with melting temperatures used to confirm product identity. Assays were variously performed on: (i) extracted DNA; (ii) cultured bacteria; (iii) urine spiked with reference strains; and (iv) bacteria harvested from clinical urines. Results were compared with those from sequencing, real-time SybrGreen PCR or phenotypic susceptibility. 

Results: Performance was similar irrespective of whether DNA, cultures or urines were used, with >90% sensitivity and specificity with respect to common β-lactamases, dfr genes and aminoglycoside resistance determinants except aadA1/A2/A3, for which carriage correlated poorly with streptomycin resistance. Fluoroquinolone-susceptible and -resistant Escherichia coli (but not other species) were distinguished by the melting temperatures of their gyrA PCR products. The time from urine to results was <3 h. 

Conclusions: The MT-PCR assays rapidly identified resistance genes from Gram-negative bacteria in urines as well as from cultivated bacteria. Used directly on urines, this assay has the potential to guide early therapy.