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.
ABSTRACT
BACKGROUND: The nexus between resistance determinants, plasmid type, and clonality appears to play a crucial role in the dissemination and survival of carbapenem-resistant Klebsiella pneumoniae (CRKP). The incidence of infections involving CRKP in Saudi Arabia is increasing and there is a need for detailed molecular profiling of this pathogen for CRKP surveillance and control. METHODS: The resistance determinants of 71 non-redundant CRKP isolates were investigated by polymerase chain reaction (PCR) and sequencing. Plasmid typing was performed using PCR-based replicon typing and the clonality of isolates was determined by multilocus sequence typing. Capsular polysaccharide synthesis genes and other virulence factors were examined using multiplex PCR. Diversity was calculated using DIVEIN, clonal relationship was determined using eBURST, and phylogenetic analysis was performed using SplitsTree4. RESULTS: A polyclonal OXA-48 gene alone was the most common carbapenemase detected in 48/71 (67.6%) isolates followed by NDM-1 alone in 9/71 (12.7%) isolates. Coproduction of OXA-48 and NDM-1 was observed in 6/71 (8.5%) isolates. Both carbapenemase genes could be transferred into an Escherichia coli recipient. CTX-M-15 was the most abundant extended-spectrum ß-lactamase gene detected in 47/71 (66.2%) isolates, whereas clone-specific CTX-M-14 (ST-199 and -709) was found in 15/71 (21%) isolates. Sixty-seven of 71 isolates were positive for one or more plasmid replicons. The replicons detected were: IncFII; IncFIIK; IncFIA; IncFIB; L/M; IncI1; and IncN. FIIK and L/M were predominant, with 69 and 67% positivity, respectively. All isolates were negative for the magA (K1), rmpA, and K2 genes and presented a non-hypermucoviscous phenotype. CONCLUSION: A polyclonal CRKP reservoir of sequence types (STs)-37, - 199, and - 152 was observed and ST-152 appeared to be a "frequent carrier" of the NDM-1 gene. ST-199, a singleton not previously reported, showed a sequence diversity suggestive of positive selection. A significant association was evident between resistance determinants and the clonal types of K. pneumoniae: all ST-152 isolates were positive for NDM-1 but negative for OXA-48; ST-199 isolates were positive for OXA-48 but negative for NDM-1; and ST-709 and -199 isolates were positive for CTX-M-14. The incidence of certain clonal types in large numbers predicts an outbreak-like situation and warrants stringent surveillance and infection control.
