Elucidation of molecular mechanism for colistin resistance among Gram-negative isolates from tertiary care hospitals.

Antimicrobial resistance is a growing concern of global public health. The emergence of colistin-resistance among carbapenem-resistant (CPR) Gram-negative bacteria causing fear of pan-resistance, treatment failure, and high mortality across the globe. AIM: To determine the genotypic colistin-resistance mechanisms among colistin-resistant (CR)Gram-negative clinical isolates along with genomic insight into hypermucoviscous(hv)-CR-Klebsiella pneumoniae. METHODS: Phenotypic colistin-resistance via broth-microdilution method. PCR-based detection of plasmid-mediated colistin resistance genes(mcr-1,2,3). Characterization of selected hvCR-K. pneumoniae via Whole-genome sequencing. RESULTS: Phenotypic colistin-resistance was 28% among CPR-Gram-negative isolates of which 90% of CR-isolates displayed MDR profile with overall low plasmid-mediated colistin resistance (mcr-2 = 9.4%;mcr-3 = 6%). Although K. pneumoniae isolates showed the highest phenotypic colistin-resistance (51%) however, relatively low plasmid-mediated gene-carriage (mcr-2 = 11.5%;mcr-3 = 3.4%) pointed toward other mechanisms of colistin-resistance. mcr-negative CR-K. pneumoniae displaying hv-phenotype were subjected to WGS. In-silico analysis detected 7-novel mutations in lipid-A modification genes includes eptA(I38V; V50L; A135P), opgE(M53L; T486A; G236S), and arnD(S164P) in addition to several non-synonymous mutations in lipid-A modification genes conferring resistance to colistin. Insertion of 6.6-kb region harboring putative-PEA-encoding gene(yjgX) was detected for the first time in K. pneumoniae (hvCRKP4771). In-silico analysis further confirmed the acquisition of not only MDR determinants but several hypervirulent-determinants displaying a convergent phenotype. CONCLUSION: overall high prevalence of phenotypic colistin resistance but low mcr-gene carriage suggested complex chromosomal mediated resistance mechanism especially in K. pneumoniae isolates. The presence of novel mutations in lipid-A modification genes and the acquisition of putative-PEA-encoding gene by hvCR-K. pneumoniae points toward the role of chromosomal determinants conferring resistance to colistin in the absence of mcr-genes.

Emergence of hypermucoviscous colistin-resistant high-risk convergent Klebsiella pneumoniae ST-2096 clone from Pakistan.

Klebsiella pneumoniae convergent clones are considered a threat to healthcare settings. Here we report a comprehensive genomic profiling of an emerging colistin-resistant K. pneumoniae ST-2096 convergent clone from Pakistan. Methods: Whole-genome sequencing was performed and raw reads were assembled antimicrobial resistance and virulence genes were predicted using various online tools. Results & conclusion: The phenotypically multidrug-resistant (MDR) and hypermucoviscous (hv) colistin-resistant K. pneumoniae (hvCRKP-10718), which, intriguingly, possessed a wide range of antimicrobial resistance (blaTEM-1A, blaOXA-1, blaOXA-232, blaCTX-M-15, blaSHV-106, oqxA, oqxB, aac(6')-Ib-cr, aadA2, aac(6')-Ib-cr, armA, tetD, mphE, msrE, fosA, dfrA1, dfrA12, dfrA14, catB3, sul1) and virulence determinants (RmpA/RmpA2, yersiniabactin [ybt], aerobactin [iuc/iut], enterobactin). Furthermore, the acquisition of various mobile genetic elements (MDR/virulent plasmids, type II integron gene cassette, insertional sequences, transposases) and associated hv capsular type made this MDR/hv isolate a convergent clone belonging to a high-risk lineage (ST-2096). Based on core-genome multilocus sequence typing and single-nucleotide polymorphism analysis, this isolate showed ≥99% nucleotide identity with MDR K. pneumoniae isolates from India, depicting its evolutionary background. This study provides a comprehensive genomic profiling of this high-risk convergent K. pneumoniae ST-2096 clone from Pakistan. Comparative genomics of MDR/hv colistin-resistant K. pneumoniae isolates with other MDR convergent strains from the Indian subcontinent indicated the emergence of this evolving superbug.