Simultaneous Genotyping Of 16S RDNA Of Nosocomial Bacteria Using High-resolution Melting Analysis

Objective To develop a real-time polymerase chain reaction coupled with high-resolution melting analysis(HRM)targeting the variable regions of 16 S rDNA gene for the genotyping of 9nosocomial bacteria including Escherichia coli,Pseudomonas aeruginosa,Klebsiella pneumoniae,Acinetobacter baumannii,Enterobacter cloacae,Staphylococcus aureus,Staphylococcus epidermidis,Enterococcus faecalis and Enterococcus Faecium.A total of 25 anonymized,clinical isolates which included in the above nine bacteria from confirmed nosocomial infection insolates were studied to validate the PCR-HRM assay.Methods Four primer pairs targeting the V1,V3,V6,V9 variable regions of 16 S rDNA were used to identify the 9 bacteria,9 isolates of standard bacterial strains were obtained to verify the 4primer pairs.The forward primer V3 PF and the reverse primer V6 PR were used as the sequencing primers.The resulting sequences were BLAST-searched against the GenBank database.PCR-HRM assay was used to detect the 4 variable regions(V1,V3,V6 and V9)of 16 S rDNA of9 bacteria,thus develop a subgroup decision-tree strategy involving Tm grouping and followed by systematic comparisons of melting shape to genotype the 9 bacteria.A total of 25 anonymized,clinical isolates from confirmed nosocomial infection insolates which included in the above 9bacteria were studied to validate the PCR-HRM assay.Results(1)The PCR results of the 4 primer pairs show that primers targeting V3,V6,V9 variable regions can be used to amplify all the 9 bacteria,primers targeting V1 variable region can only amplify Staphylococcus aureus,Staphylococcus epidermidis,Enterococcus faecalis and Enterococcus Faecium species;The 9 reference strains used in the study have 99% to 100%similarity for their type strain in Genbank.(2)The differentiation of 9 nosocomial bacteria wasachieved using a multiparameter,decision-tree approach that was based on grouping according to melting temperature,and sequential comparisons of melting profiles in the V1,V3,V6 and V9 variable regions of 16 S rDNA: 1)The initial classification of the nine bacteria present in nosocomial infection was based on Gram typing.Organisms were allocated into one of two groups based on the specific PCR of V1 region;2)Gram-positive species including Staphylococcus aureus,Staphylococcus epidermidis,Enterococcus faecalis and Enterococcus Faecium.The differences in Tm between Staphylococcus(including Staphylococcus aureus and Staphylococcus epidermidis;mean Tm,88.20 ?)and Enterococcus(Enterococcus faecalis and Enterococcus Faecium;mean Tm,89.75?)of Gram-positive species in V9 region were highly significant.Then we differentiate Staphylococcus aureus from Staphylococcus epidermidis in V3 region.Staphylococcus aureus and Staphylococcus epidermidis had clearly distinguishable melting curves and could be differentiated through direct visualization.V1 region was used to differentiate Enterococcus faecalis(Tm,85.74?)and Enterococcus Faecium(Tm,86.94?),they had clearly distinguishable melting peaks in this region;3)Gram-negative species including Escherichia coli,Pseudomonas aeruginosa,Klebsiella pneumoniae,Acinetobacter baumannii and Enterobacter cloacae,they were classified into three groups in V3 region based on differences in Tm and the melting curve.Group 1 consisted of Klebsiella pneumoniae and Acinetobacter baumannii.Group 2 consisted of Escherichia coli and Enterobacter cloacae.Group 3 only including one species of Pseudomonas aeruginosa.Because of the clearly distinguishable melting peaks and melting curve,Pseudomonas aeruginosa can be identified from the other four Gram-negative species directly in V3 region.Klebsiella pneumoniae(Tm,86.20?)and Acinetobacter baumannii(Tm,84.84?)of Group 1 can differentiated in V6 region they had clearly distinguishable melting peaks in this region.Escherichia coli and Enterobacter cloacae of Group 2 are located in the same domain and have similar melting profiles in V3,V6,V9 regions,making it very difficult to distinguish directly through melting curve analysis.By constructing a heteroduplex formation using Escherichia coli as a reference,the two species can be differentiated from each other.(3)The results of real-time PCR-HRM identification versus microbiological culture for 25 consecutiveclinical isolates from patients with confirmed nosocomial infection showed that 22 isolates were correctly identified by PCR-HRM assay.Of the remaining 3 isolates,1 isolate was incorrectly identified(HRM identified as Staphylococcus epidermidis,Microbiological culture identified as Staphylococcus aureus),2 isolates were not included in the subgroup decision-tree,and these 3isolates were classified as unidentified by the HRM assay.Conclusion We developed a PCR-HRM subgroup decision-tree approach to identify nine nosocomial bacteria;PCR-HRM identification versus microbiological culture for 25 consecutive clinical isolates from patients with confirmed nosocomial infection showed that 88%(22/25)were correctly identified by PCR-HRM assay.