Carbapenemase-Encoding Genes in Pseudomonas aeruginosa: Multiplex PCR Detection, Microarray Based Detection and Gene Sequencing

Abstract

Background: In 2017 the World Health Organization (WHO) classified carbapenem-resistant Pseudomonas among the most critical multidrug-resistant bacteria for urgent attention.   Aims: Evaluation of two molecular methods for detection of carbapenemase encoding genes in carbapenem resistant Pseudomonas aeruginosa (CRPA), and alignment of detected gene sequences with gene bank data. Materials and Methods: Twenty two previously detected CRPA isolates were tested for bacterial identification and antibiotic sensitivity by verigene-nanosphere technique. Verigene-nanosphere microarray based assay and multiplex PCR were tested for detection of carbapenem encoding genes. Carbapenemase encoding genes sequencing of parent strain and its UV mutant followed by nucleotide sequence alignments were conducted. Bacterial resistance to carbapenem was tested after induced plasmid curing by sodium dodecyl sulfate (SDS) and heat. Results: Typical results of conventional PCR were obtained using multiplex PCR for a set of genes, including IMP, VIM, SME. Verigene-nanosphere detected 3, 0, 0 among 5, 4, 11 known positive strains for VIM, IMP, KPC genes respectively. Carbapenem resistance was preserved after treating CRPA strain by heat or SDS. The nucleotide sequence alignments of VIM, IMP and KPC genes showed relatedness with many Gram negative species. VIM gene was lost in UV mutant and the IMP and KPC genes were preserved, but 1-2% sequence modification occurred without a change in resistance to imipenem and meropenem. Conclusion: The developed multiplex PCR detected successfully a set of carbapenem encoding genes, while microarray based verigene-nanosphere failed to detect most of the genes under the current experimental conditions. The nucleotide alignments of VIM, IMP and KPC genes revealed that these gene sequences are distributed among species of Gram negative bacteria. IMP and KPC genes were preserved in UV mutant with no damage, repaired damage or little modification of the nucleotide sequence and meanwhile carbapenem resistance was preserved.