Development of an optimized random amplified polymorphic DNA protocol for fingerprinting of Klebsiella pneumoniae

Abstract

Aims: To develop an optimized random amplified polymorphic DNA (RAPD) protocol for fingerprinting clinical isolates of Klebsiella pneumoniae. Methods and Results: Employing factorial design of experiments, repeatable amplification patterns were obtained for 54 nosocomial isolates using 1 μmol 1−1 primer, 4 mmol 1−1 MgCl2, 0·4 mmol 1−1 dNTPs, 2·5 U Taq DNA polymerase and 90 ng DNA template in a total volume of 25 μl. The optimum thermocycling program was: initial denaturation at 94°C for 4 min followed by 50 cycles of 1 min at 94°C, 2 min at 34°C, 2 min at 72°C and a final extension at 72°C for 10 min. The optimized RAPD protocol was highly discriminatory (Simpson’s diversity index, 0·982), and all isolates were typable with repeatable patterns (Pearson’s similarity coefficient ∼100%). Seven main clusters were obtained on a similarity level of 70% and 32 distinct clusters on a similarity level of 85%, reflecting the heterogeneity of the isolates. Conclusions: Systematic optimization of RAPD generated reliable DNA fingerprints for nosocomial isolates of K. pneumoniae. Significance and Impact of the Study: This is the first report on RAPD optimization based on factorial design of experiments for discrimination of K. pneumoniae.

Correlation of Multi-drug Resistance, Integron and blaESBL Gene Carriage With Genetic Fingerprints of Extended-Spectrum β-Lactamase Producing Klebsiella pneumoniae

“…Genetic profiles of the isolates by RAPD, confirmed by PFGE have been reported in our previous article (7). Presence of bla ESBL genes ( bla SHV , bla TEM and bla CTX-M ) and the sequencing result for the isolates were also previously reported (9).…”

“…Genetic profiles of the isolates by RAPD (Figure 2) which were confirmed by PFGE, showed six major clusters (a-f) on a similarity level of 70%, and 21 different groups on a similarity level of 85% (7). Characterization of bla ESBL genes from our previous work showed that 27 isolates (77.1%) harbored bla SHV genes including bla SHV-12 , bla SHV-5 and bla SHV-11 , 17 (48.6%) carried bla TEM genes characterized as bla TEM-1 by sequencing, 16 (45.71%) carried bla CTX-M-I which belonged to bla CTX-M-15 and 10 (28.57%) contained bla CTX-M-III characterized as bla CTX-M-8 (9).…”

“…Various typing methods have been applied to understand transmission patterns of resistance genes and management of nosocomial infections (6). We have previously developed an optimized RAPD-PCR protocol for genotyping K. pneumoniae , comparable to PFGE (7). To understand the associations between phenotypic and genetic characteristics of multi-drug resistant pathogens can be useful for reliable detection of these bacteria in epidemiological studies.…”

Detection of a CTX-M group 2 beta-lactamase gene in a Klebsiella pneumoniae isolate from a tertiary care hospital, Trinidad and Tobago

“…RAPD-PCR was chosen because it is a rapid and simple method which when optimized has proven competitive with the gold standard of pulse field gel electrophoresis [26, 27]. RAPD1, also referred to as Primer 640 by other authors [13, 26], proved to be optimal for DNA fingerprinting since it allowed the clear distinction of DNA banding patterns for all of the isolates tested. The resulting RAPD profiles for this select group of isolates showed that there was diversity among those isolated during 2008–2010 and those recovered in 2015.…”

“…Samples were initially screened for RAPD typing using five different primers: RAPD1 (5′-CGTGGGCCT), RAPD2 (5′-TCGTCGGCGT), RAPD3 (5′-GTGACGTAGG), RAPD4 (5′-CTTGAGTGGA), RAPD5 (5′-GAGATGACGA) (Sigma-Aldrich). RAPD–PCR was conducted under reaction conditions described by Ashayeri-panah et al [13]. The amplified products were separated by electrophoresis in a 2.0% agarose gel containing GelRed™ run in 1 × TAE buffer at 65 V for 3 h 30 min until amplified fragments are separated.…”