ABSTRACT
Background: Hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has recently aroused an extremely severe health challenge and public concern. However, the underlying mechanisms of fitness costs that accompany antibiotic resistance acquisition remain largely unexplored. Here, we report a hv-CRKP-associated fatal infection and reveal a reduction in virulence due to the acquisition of aminoglycoside resistance. Methods: The bacterial identification, antimicrobial susceptibility, hypermucoviscosity, virulence factors, MLST and serotypes were profiled.The clonal homology and plasmid acquisition among hv-CRKP strains were detected by XbaI and S1-PFGE. The virulence potential of the strains was evaluated using Galleria mellonella larvae infection model, serum resistance assay, capsular polysaccharide quantification, and biofilm formation assay. Genomic variations were identified using whole-genome sequencing (WGS). Results: Four K. pneumoniae carbapenemase (KPC)-producing CRKP strains were consecutively isolated from an 86-year-old patient with severe pneumonia. Whole-genome sequencing (WGS) showed that all four hv-CRKP strains belonged to the ST11-KL64 clone. PFGE analysis revealed that the four ST11-KL64 hv-CRKP strains could be grouped into the same PFGE type. Under the pressure of antibiotics, the antimicrobial resistance of the strains increased and the virulence potential decreased. Further sequencing, using the Nanopore platform, was performed on three representative isolates (WYKP586, WYKP589, and WYKP594). Genomic analysis showed that the plasmids of these three strains underwent a large number of breaks and recombination events under antibiotic pressure. We found that as aminoglycoside resistance emerged via acquisition of the rmtB gene, the hypermucoviscosity and virulence of the strains decreased because of internal mutations in the rmpA and rmpA2 genes. Conclusion: This study shows that ST11-KL64 hv-CRKP can further evolve to acquire aminoglycoside resistance accompanied by decreased virulence to adapt to antibiotic pressure in the host.
ABSTRACT
Antibiotic resistance (AMR) has always been a hot topic all over the world and its mechanisms are varied and complicated. Previous evidence revealed the metabolic slowdown in resistant bacteria, suggesting the important role of metabolism in antibiotic resistance. However, the molecular mechanism of reduced metabolism remains poorly understood, which inspires us to explore the global proteome change during antibiotic resistance. Here, the sensitive, cotrimoxazole-resistant, amikacin-resistant, and amikacin/cotrimoxazole -both-resistant KPN clinical isolates were collected and subjected to proteome analysis through liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). A deep coverage of 2,266 proteins were successfully identified and quantified in total, representing the most comprehensive protein quantification data by now. Further bioinformatic analysis showed down-regulation of tricarboxylic acid cycle (TCA) pathway and up-regulation of alcohol metabolic or glutathione metabolism processes, which may contribute to ROS clearance and cell survival, in drug-resistant isolates. These results indicated that metabolic pathway alteration was directly correlated with antibiotic resistance, which could promote the development of antibacterial drugs from "target" to "network." Moreover, combined with minimum inhibitory concentration (MIC) of cotrimoxazole and amikacin on different KPN isolates, we identified nine proteins, including garK, uxaC, exuT, hpaB, fhuA, KPN_01492, fumA, hisC, and aroE, which might contribute mostly to the survival of KPN under drug pressure. In sum, our findings provided novel, non-antibiotic-based therapeutics against resistant KPN.
ABSTRACT
BACKGROUND: The convergence of carbapenem-resistance and hypervirulence in Klebsiella pneumoniae has led to a significant public health challenge. In recent years, there have been more and more reports on carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) isolates. MATERIALS AND METHODS: Clinical data of patients infected with CR-hvKP from January 2019 to December 2020 in a tertiary hospital were retrospectively evaluated. The number of isolates of Klebsiella pneumoniae, hypermucoviscous Klebsiella pneumoniae (hmKP), carbapenem-resistant hypermucoviscous Klebsiella pneumoniae (CR-hmKP) and carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) collected during the period of 2 years was calculated. The antimicrobial resistance gene, virulence-associated gene, capsular serotype gene and multilocus sequence typing (MLST) of CR-hvKP isolates were detected by PCR. RESULTS: During the study period, a total of 1081 isolates of non-repeat Klebsiella pneumoniae were isolated, including 392 isolates of hypermucoviscous Klebsiella pneumoniae (36.3%), 39 isolates of CR-hmKP (3.6%), and 16 isolates of CR-hvKP (1.5%). About 31.2% (5/16) of CR-hvKP were isolated from 2019, and 68.8% (11/16) of CR-hvKP were isolated from 2020. Among the 16 isolates of CR-hvKP, 13 isolates were ST11 and serotype K64, 1 isolate was ST11 and serotype K47, 1 isolate was ST23 and serotype K1, and 1 isolate was ST86 and serotype K2. The virulence-associated genes entB, fimH, rmpA2, iutA, iucA were present in all of 16 CR-hvKP isolates, followed by mrkD (n=14), rmpA (n=13), aerobactin (n=2), allS (n=1). Sixteen CR-hvKP isolates all carry carbapenemase gene bla KPC-2 and extended-spectrum ß-lactamase gene bla SHV. ERIC-PCR DNA fingerprinting results showed that 16 CR-hvKP isolates were highly polymorphic, and there were significant differences in bands among the isolates, presenting a sporadic state. CONCLUSION: Although CR-hvKP was sporadically distributed, it showed an increasing trend year by year. Therefore, clinical attention should be paid, and necessary measures should be taken to avoid the cloning and transmission of superbacterium CR-hvKP.
