Insertion element mediated mgrB disruption and presence of ISKpn28 in colistin-resistant Klebsiella pneumoniae isolates from Saudi Arabia

Abstract

Background: In Klebsiella pneumoniae, mgrB and components of pmrHFIJKLM operon play a major role in colistin resistance.

Methods: We analyzed 23 nonduplicating colistin-resistant K. pneumoniae isolates, collected during the years 2011-2015, for the possible mechanism underlying their nonsusceptibility to colistin. Isolates were tested for their minimum inhibitory concentrations and antibiotic resistance determinants and genotyped by multilocus sequence typing (MLST). The MLST genes, antibiotic-resistant genes, and the genes of two component system (TCS), including mgrB, PhoQ/PhoP, pmrA/B, and CrrAB, were investigated by PCR amplification and Sanger sequencing.

Results: All isolates were distributed in eight sequence types (STs) and showed mutations either in mgrB or PhoP genes. ISKpn14 was found in 10, ISKpn28 in four, and IS903 in three isolates. One isolate showed deletion of a single nucleotide in mgrB open reading frame causing premature stop codon. L26Q substitution in PhoP was found in five isolates.

Conclusion: The mutations in mgrB were mostly mediated by insertion elements (IS). ISKpn14 is the major IS while ISKpn28 is reported for the first time in mediating mgrB disruption. IS903, an IS5 family member, involved in mgrB disruption in three ST-152 NDM-1-positive isolates, was previously responsible for omp-36 disruption in our carbapenem-resistant K. pneumoniae and appears to contribute to transform the isolates into a pan-drug ones. Also, the abundance of insertion sites in mgrB indicates the plasticity of this gene. In our isolates, IS-mediated colistin resistance appears to be a later phenomenon than mutation in PhoP gene.

Keywords: Klebsiella pneumoniae; colistin resistance; insertion elements; mgrB gene mutations.

Figure 1

(A) mgrB gene sequence with the three ISs inserted at different positions in the open reading frame and the promoter region of the gene. The bold-face TTTTGA and TAAACT are −35 and −10 boxes of the promoter, whereas GTG and TAA are start and stop codons, respectively. The arrows and the numbers at their base indicate the orientation and the upstream (−) and downstream (+) positions of IS from the start codon. The numbers in parentheses indicate the multiples of isolates with the particular sequence type; (B) Phylogenetic relationship based on comparisons of IS903 insert sequence in omp-36 gene of our 2010 outbreak isolates in and the mgrB gene of the present study population and six other GenBank depositions; (C) Phylogenetic relationship based on ISKpn14 sequence among isolates of this study carrying the insert. The bar in (B) and (C) is the divergence scale denoting number of substitutions per site. Abbreviations: IS, insertion element; KP, Klebsiella pneumoniae; ST, sequence type.