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Characterization of phage resistance and phages capable of intestinal decolonization of carbapenem-resistant Klebsiella pneumoniae in mice.

ABSTRACT: Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as a severe global health challenge. We isolate and characterize two previously unidentified lytic phages, P24 and P39, with large burst sizes active against ST11 KL64, a major CRKP lineage. P24 and P39 represent species of the genera Przondovirus (Studiervirinae subfamily) and Webervirus (Drexlerviridae family), respectively. P24 and P39 together restrain CRKP growth to nearly 8 h. Phage-resistant mutants exhibit reduced capsule production and decreased virulence. Modifications in mshA and wcaJ encoding capsule polysaccharide synthesis mediate P24 resistance whilst mutations in epsJ encoding exopolysaccharide synthesis cause P39 resistance. We test P24 alone and together with P39 for decolonizing CRKP using mouse intestinal colonization models. Bacterial load shed decrease significantly in mice treated with P24 and P39. In conclusion, we report the characterization of two previously unidentified lytic phages against CRKP, revealing phage resistance mechanisms and demonstrating the potential of lytic phages for intestinal decolonization.

SUBMITTER: Fang Q

PROVIDER: S-EPMC8758719 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Characterization of phage resistance and phages capable of intestinal decolonization of carbapenem-resistant Klebsiella pneumoniae in mice.

Fang Qingqing Q Feng Yu Y McNally Alan A Zong Zhiyong Z

Communications biology 20220113 1

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as a severe global health challenge. We isolate and characterize two previously unidentified lytic phages, P24 and P39, with large burst sizes active against ST11 KL64, a major CRKP lineage. P24 and P39 represent species of the genera Przondovirus (Studiervirinae subfamily) and Webervirus (Drexlerviridae family), respectively. P24 and P39 together restrain CRKP growth to nearly 8 h. Phage-resistant mutants exhibit reduced capsule prod ...[more]

PMID: 35027665

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Project description:We isolated and characterized a novel phage from hospital sewage, P13, able to lyse ST11 K47 carbapenem-resistant Klebsiella pneumoniae (CRKP), a major CRKP lineage. P13 formed a large lytic plaque (3.0 to 6.0 mm in diameter) in double-layer LB agar after overnight coculture with its host bacterial strain. A translucent halo formed when the culture was prolonged to 48 h. P13 showed a narrow host range only lysing ST11 K47 CRKP with a burst size of around 167 PFU/cell and exhibited broad pH and thermal stability. Genome sequencing showed that P13 contains no virulence, lysogenic or antimicrobial resistance genes, making this lytic phage a potential agent for phage therapy. Transmission electron microscopy showed that P13 exhibited typical morphology of the family Podoviridae with an isometric head and a short noncontracted tail. Genomic analysis showed that P13 belongs to a novel species of the genus Przondovirus, subfamily Studiervirinae, family Autographiviridae. P13-resistant mutants of bacteria emerged after 4 h exposure to the phage. Interruptions of wbaP (encoding capsule polysaccharide synthesis) by insertion sequence IS903B mediated P13 resistance. The rapid emergence of resistant mutants represents a disadvantage for P13 as a therapeutic phage and highlights the need for recovery of a range of phages binding to different surface receptors of host bacteria to further extend their utility as a potential tool against CRKP. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major challenge for infection control and clinical management. Alternative therapies to antimicrobial agents are urgently needed and bacteriophages (phages) are an attractive option. However, more novel lytic phages and more studies to reveal phage-resistant mechanisms are needed to overcome phage resistance. In this study, we isolated and characterized a novel species of lytic phage active against CRKP. We found this phage exhibited delayed formation of halo, which is atypical compared to other characterized similar phages, and we provide an explanation for this phenotype based on genomic analysis. We also identified mechanisms mediating resistance to the phage.

Project description:Carbapenem-resistantKlebsiella pneumoniae(CR-Kp) is significant threat to public health worldwide. The primary reservoir for CR-Kp is the intestinal tract, where the bacterium is usually present at low density, but can bloom following antibiotic treatment, mostly in hospital settings. The impact of disturbances in the intestinal environment on the fitness, survival, expansion, and drug susceptibility of this pathogen is not well-understood. Nevertheless, gaining such knowledge could lead to innovative intervention strategies for addressing colonization and infection. Here, we adopted anin vivomodel to examine the transcriptional adaptation of a CR-Kp clinical isolate to immune activation in the intestine. We report that as early as 6 hours following host treatment with anti-CD3 antibody, CR-Kp underwent rapid transcriptional changes including downregulation of genes involved in sugar utilization and amino acid biosynthesis, and upregulation of genes involved in amino acid uptake and catabolism, antibiotic resistance, and stress response. In agreement with these findings, the concentration of oxidative species and amino acids was increased in the mouse intestine following treatment. Genes encoding for proteins containing the domain of unknown function (DUF) 1471 were particularly upregulated, however their deletion did not impair CR-Kp fitness in vivoupon immune activation. Transcription factor enrichment analysis identified the global regulator cAMP-Receptor Protein CRP as a potential orchestrator of the observed transcriptional signature. In keeping with the recognized role of CRP in regulating utilization of alternative carbon sources, CRP deletion in CR-Kp resulted in strongly impaired gut colonization, but this effect was not amplified by immune activation. Thus, following intestinal colonization, which occurs in a CRP-dependent manner, CR-Kp can rapidly respond to immune cues by implementing a well-defined and complex transcriptional program whose direct relevance towards bacterial fitness remains cryptic. Further analyses utilizing this model may identify key factors to tackle the intestinal stage of CR-Kp colonization.

Project description:Background: The rate of carbapenem-resistant Klebsiella pneumoniae (CRKP) infection has been increasing rapidly worldwide and, poses a significant risk to human health. Effective methods are urgently needed to address treatment failures related to antibiotic resistance. Recent research has reported that some drugs in combination with antibiotics have displayed synergistic killing of resistant bacteria. Here, we investigated whether glutathione (GSH) can synergize with meropenem, and enhance its effectiveness against CRKP. Methods: Synergistic activity was assessed by checkerboard and time-killing assays. The mechanism of these combinations was assessed by total ROS and membrane permeability assays. The bacterial metabolites were assessed by LC‒MS/MS. Results: The FICIs of GSH and meropenem were approximately 0.5 and the combined treatment with GSH and meropenem resulted in a more than 2log10 CFU/mL reduction in bacteria compared to the individual treatments. These findings indicated the synergistic effect of the two drugs. Moreover, the meropenem MIC of CRKP was reduced to less than 4 mg/L when combined with 6 mg/mL GSH, indicating that GSH could significantly reverse resistance to meropenem in bacteria. The production of ROS in bacteria was determined by flow cytometry. After adding GSH, the ROS in the GSH group and the combined group was significantly higher than that in the control and meropenem groups, but there was no significant difference between the combined and GSH groups. The metabolic disturbance caused by GSH alone and in combination with meropenem was significant intracellularly and extracellularly, especially in terms of glycerophospholipid metabolism, indicating that the synergistic effect of the combined use of GSH and meropenem was relevant to glycerophospholipid metabolism. In addition, we measured the cell membrane permeability. The cell membrane permeability of the combination group was significantly higher than that of the blank control or monotreatment groups. This confirmed that the GSH can serve as a meropenem enhancers by disturbing glycerophospholipid metabolism and increasing cell membrane permeability. Conclusion: GSH and meropenem display a synergistic effect, wherein GSH increases the sensitivity of CRKP to meropenem. The synergy and susceptibility effects are thought to related to the increased membrane permeability resulting from the perturbations in glycerophospholipid metabolism, presenting a novel avenue for CRKP treatment.

Project description:Carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a critical public health threat. However, the association between intestinal colonization and parenteral infection among pediatric patients has not been elucidated. We collected 8 fecal CRKP strains and 10 corresponding CRKP strains responsible for extraintestinal infection from eight patients who did not manifest infection upon admission to the hospital. Paired isolates showed identical resistance to antimicrobials and identical virulence in vitro and in vivo. wzi capsule typing, multilocus sequence typing, and whole-genome sequencing (WGS) indicated high similarity between paired colonizing and infecting isolates. Mutations between colonizing and infecting isolate pairs found by WGS had a distinctive molecular signature of a high proportion of complex structural variants. The mutated genes were involved in pathways associated with infection-related physiological and pathogenic functions, including antibiotic resistance, virulence, and response to the extracellular environment. The latter is important for bacterial infection of environmental niches. Various mutations related to antibiotic resistance, virulence, and colonization that were not associated with any particular mutational hot spot correlated with an increased risk of extraintestinal infection. Notably, novel subclone carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) KL19-ST15 exhibited hypervirulence in experimental assays that reflected the severe clinical symptoms of two patients infected with the clonal strains. Taken together, our findings indicate the association between CRKP intestinal colonization and extraintestinal infection, suggesting that active screening for colonization on admission could decrease infection risk in children. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae (CRKP) causes an increasing number of nosocomial infections, which can be life-threatening, as carbapenems are last-resort antibiotics. K. pneumoniae is part of the healthy human microbiome, and this provides a potential advantage for infection. This study demonstrated that CRKP intestinal colonization is strongly linked to extraintestinal infection, based on the evidence given by whole-genome sequencing data and phenotypic assays of antimicrobial resistance and virulence. Apart from these findings, our in-depth analysis of point mutations and chromosome structural variants in patient-specific infecting isolates compared with colonizing isolates may contribute insights into bacterial adaptation underlying CRKP infection. In addition, a novel subclone of carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) was observed in the study. This finding highlights the importance of CRKP active surveillance among children, targeting in particular the novel high-risk CR-hvKP clone.

Dataset Information

Characterization of phage resistance and phages capable of intestinal decolonization of carbapenem-resistant Klebsiella pneumoniae in mice.

Publications

Characterization of phage resistance and phages capable of intestinal decolonization of carbapenem-resistant Klebsiella pneumoniae in mice.

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