Use of rep-PCR for Molecular Genotyping and Inventory among Nosocomial Infection Associated Isolates

Background: Nosocomial infection has emerged as a global challenge for healthcare professionals. Identifying the source of infection is an important task for hospitals to develop a cost-effective strategy for prevention. Molecular subtyping is a powerful tool for determining the source of infection by their genetic relatedness among isolates. However, the conventional method (ie. pulsed-field gel electrophoresis), is time consuming and skill intensive for interpretable results. Currently repetitive-sequence-based PCR (rep-PCR) technique has been developed to an automated procedure that may provide a rapid method for molecular subtyping of bacteria. Such rapid method provides same-day genotyping results and enables infection control practitioners to timely eliminate further nosocomial AB transmission. The reproducibility and digital output of rep-PCR also provide the opportunity for hospitals to inventory isolate patterns.

Objective: To evaluate the feasibility of using rep-PCR for nosocomial infection associated isolate typing.

Methods: Twelve Staphylococcus aureus, 19 Acinetobacter baumannii, and 12 Legionella pneumophila isolates were used in this study. A rep-PCR assay and a capillary electrophoresis system (Bioanalyzer 2100, Agilent) were used for molecular analysis. We followed the standardized procedures for analysis including: (1) extract DNA from the isolated cultures; (2) amplify samples using rep-PCR and the specie-specific, in-house typing kits; (3) separate fragments via electrophoresis performed in a microfluidics DNA LabChip; and (4) analyze data using dendrogram, similarity matrix, and overlay analysis.

Results: (1) the patterns of the 12 S. aureus isolates are distinctive by rep-PCR, same to PFGE typing; (2) the patient isolates of A. baumannii (Key 4, 5, 7, 8, 9, 10) are the same clone and epidemiologically linked to one environmental isolate (Key 6) isolated from hospital potable water; (3) A patient isolate of L. pneumophila matches the isolates cultured from the patient room faucet. Healthcare facility can build an isolate database based on rep-PCR and capillary electrophoresis results which can be used to determine the possible source of infection in future outbreak investigation. In-house rep-PCR protocol/reagents/condition and generic labchip may be a cost-effective alternative compatible to the brand name kits and chips, which can reduce the cost per isolate significantly.

Conclusions: The signal-base electrophoresis by rep-PCR provide more reproducible results than the analog image-base results by PFGE. Times to results were 6 to 8 hr for rep-PCR compared to 3 to 5 days for PFGE. Rapid, standardized results and high reproducibility make the rep-PCR a valuable tool for use in nosocomial transmission investigations.