Automated laser fluorescence analysis of randomly amplified polymorphic DNA: A rapid method for investigating nosocomial transmission of Acinetobacter baumannii

Abstract

Summary.A rapid method for genotyping Acinetobacter baumannii based on PCRfingerprinting with fluorescent primers was evaluated. Automated laser fluorescence analysis (ALFA) enabled on-line generation of high resolution DNA-fingerprints during polyacrylamide gel electrophoresis of randomly amplified polymorphic DNA (RAPD) products. The results were in concordance with macro-restriction fragment patterns produced by pulsed-field gel electrophoresis (PFGE) of ApaI digests of chromosomal DNA. RAPD-ALFA was able to identify homologous strains suggestive of horizontal transmission in < 8 h after colonies were obtained on solid media, whereas PFGE analysis took c. 90 h. Speed and digitised data format renders RAPD-ALFA attractive for routine in-house epidemiological screening of isolates from intensive care and other hospital units.

Population structure and antibiotic resistance of Acinetobacter DNA group 2 and 13TU isolates from hospitals in the UK

“…As reported previously [4][5][6], RAPD fingerprinting was found to be a reliable and reproducible typing technique for Acinetobacter DNA group 2 and 13TU isolates. It is important to note that the highly standardised methodology [13] employed in this study is essential to achieve the necessary level of reproducibility with the RAPD technique.…”

“…Each batch of RAPD experiments included strain RUH 2037 as a reproducibility standard, as well as a negative control containing all components except template DNA. A cut-off value of >72% similarity -shown previously in similar RAPD experiments to distinguish between unrelated genotypes of A. baumannii [5,13] -was used to differentiate clusters of unrelated isolates. Clustering of molecular size markers (100-bp ladder) was used as a measure of the level of inherent variability following merging and clustering of data from different gels.…”

“…Randomly amplified polymorphic DNA (RAPD) fingerprinting was chosen because it has been shown to be particularly useful and reproducible with isolates of Acinetobacter spp. [4][5][6]. The study also aimed to correlate the population structure with antibiotic resistance data to determine whether specific resistances were associated with the ability of particular strains to cause outbreaks.…”

PCR-based DNA fingerprinting (REP-PCR, AP-PCR) and pulsed-field gel electrophoresis characterization of a nosocomial outbreak caused by imipenem- and meropenem-resistant Acinetobacter baumannii

“…baumannii complex, concluded that both techniques are useful for molecular typing. Also, Grundmann et al [19], investigating the nosocomial transmission of A. baumannii , concluded that PCR‐fingerprinting results were consistent with macrorestriction fragment patterns obtained by PFGE.…”

Detection of cross-transmission of multiresistant Gram-negative bacilli and Staphylococcus aureus in adult intensive care units by routine typing of clinical isolates

“…The present study was set up to describe the potential of routine typing of bacterial pathogens that are frequently involved in nosocomial infections, with the aim of early identification of possible cross‐transmissions, and to compare this approach with conventionally employed discrimination techniques (antibiograms). The typing was performed by PCR fingerprinting using random amplification of polymorphic DNA (RAPD), coupled with automated laser fluorescence analysis (ALFA), which allows the generation of digital PCR fingerprint data of high accuracy [9]. With this approach, the strain frequency of multidrug‐resistant Gram‐negative bacteria (MR GNB) and Staphyhcoccus aureus was continuously investigated in two adult ICUs over a period of 12 months.…”