Molecular Mechanism of Polymyxin Resistance in Multidrug-Resistant Klebsiella pneumoniae and Escherichia coli Isolates from Henan Province, China: A Multicenter Study

Abstract

To evaluate polymyxin-resistant Klebsiella pneumoniae and Escherichia coli prevalence and characteristics in the Henan province, China. Materials and Methods: A total of 2301 bacterial isolates collected at six hospitals were assessed. Their response to polymyxin was evaluated by minimum inhibitory concentration (MIC) analysis, and the mobilized colistin resistance (mcr) and carbapenemase gene were explored. Mutations on mgrB, phoPQ, pmrAB, and crrAB in polymyxin-resistant K. pneumoniae were detected by PCR. phoP, phoQ, pmrK, pmrA, pmrB, and pmrC transcriptional levels were quantified by RT-qPCR. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing were performed to determine the phylogenetic relationship between the polymyxin-resistant isolates. Results: Of the E. coli and K. pneumoniae isolates identified, 0.3% and 1.4% were polymyxin-resistant, respectively, with MICs of 4-64 μg/mL. All polymyxin-resistant isolates were susceptible to tigecycline. Four E. coli isolates were mcr-1-positive and one was carbapenem-resistant, carrying bla NDM-5 and mcr-1. One K. pneumoniae isolate was mcr-1-positive and nine were carbapenem-resistant (PRCRKP), carrying bla KPC-2 but not mcr-1.The five E. coli isolates belonged to four sequence types (ST2, ST132, ST632, and ST983). All PRCRKP isolates belonged to ST11. However, all 16 isolates belonged to different PFGE types with <95% genetic similarity. Insertion sequences in mgrB were detected in nine (81.8%) polymyxin-resistant K. pneumoniae samples. Colistin resistance was linked with pmrHFIJKLM operon upregulation, with phoP, phoQ, and pmrK being overexpressed in all but one of the polymyxin-resistant K. pneumoniae samples. Furthermore, 33.3% of patients carrying polymyxin-resistant isolates had previously used polymyxin, and 66.7% patients displayed good clinical outcomes. Conclusion:The K. pneumoniae polymyxin resistance rate was slightly higher than that of E. coli and mcr-1 was more common in E. coli than in K. pneumoniae. Moreover, the insertion of ISkpn14 into mgrB may be the main contributor to polymyxin-resistance in K. pneumoniae in Henan.

Plasmids as donors of Insertion Sequence elements mediating colistin resistance in Klebsiella pneumoniae

“…In this scenario, there may be few barriers to prevent inducible colistin resistance among bacterial isolates encoding ISKpn25 on their plasmids.In contrast, IS elements ISKpn14 and ISKpn26 are most often identified from clinical samples from China. These results agree with recent molecular analyses which reveal the presence of ISKpn14 as the principle IS element disrupting the mgrB gene of colistin resistant clinical K. pneumoniae isolates from 6 hospitals across China(Yan et al, 2021). ISKpn26 is associated with IncFII or IncFII and IncR fusion plasmids.…”

“…Across two independent samples of 31 and 49 clinically isolated colistin resistant K. pneumoniae from Taiwan, 34.7% (n=17/49), and 32.2% (n=10/31) carried IS elements in the mgrB gene (Yang et al, 2020;Berglund et al, 2018), while 30% (n=6/20) clinical colistin resistant K. pneumoniae isolates from Iran carried IS elements insertion, either IS5-like or IS1-like in mgrB (Haeili et al, 2017). More strikingly, from a sample of 11 clinical colistin resistant K. pneumoniae isolates from China, 9 carried IS elements in the mgrB gene (Yan et al, 2021), while 93.75% (n=15/16) clinical isolates from Greece harboured ISKpn26-like element disruption in mgrB (Zhu et al, 2019). ISs elements including ISKpn25, ISKpn26, IS903B and ISKpn14 are common IS elements targeting genes involved in colistin resistance (Haeili et al, Further evidence for the involvement of plasmids as donors for IS elements is provided by a recent cloning assay.…”

“…This may present a serious clinical threat, as selective colistin pressure in the hospital environment has been linked to the genetic transposition of IncFII and IncR fusion plasmids, and IncR plasmids are the most commonly identified plasmid Inc groups from ISKpn14 carrying plasmids, suggesting these plasmid Inc groups are receptive towards IS element uptake and maintenance. In addition, both ISKpn26 and ISKpn14 containing plasmids also carried a high percentage of carbapenem resistant genes, a feature also detected in ISKpn14/ ISKpn26 mgrB disrupted K. pneumoniae isolates from China(Yan et al, 2021). This may represent a worrisome clinical threat, based on evidence that CPKP isolates are successful in clinical settings.…”

“…IS elements in K. pneumoniae(Berglund et al, 2018;Yang et al, 2020;Yan et al, 2021). In this scenario, there may be few barriers to prevent inducible colistin resistance among bacterial isolates encoding ISKpn25 on their plasmids.In contrast, IS elements ISKpn14 and ISKpn26 are most often identified from clinical samples from China.…”

Development of Single Nucleotide Polymorphism (SNP)-Based Triplex PCR Marker for Serotype-specific Escherichia coli Detection

“…However, the differentiation of bacteria at the serovar level remains challenging, and SNP-based approaches for differentiating specific strains have been applied to various bacterial species, such as Escherichia [16,17], Salmonella [18], Brucella [19,20], and Bacillus species [21]. To date, several molecular methods have been used for the detection of E. coli [3,10,11,17,22,23]. However, SNP-based techniques have become increasingly attractive for the efficient detection of E. coli compared to other recognized molecular techniques [13].…”