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Identifying virulence determinants of multidrug-resistant Klebsiella pneumoniae in Galleria mellonella.

ABSTRACT: Infections caused by Klebsiella pneumoniae are a major public health threat. Extensively drug-resistant and even pan-resistant strains have been reported. Understanding K. pneumoniae pathogenesis is hampered by the fact that murine models of infection offer limited resolution for non-hypervirulent strains which cause the majority of infections. The insect Galleria mellonella larva is a widely used alternative model organism for bacterial pathogens. We have performed genome-scale fitness profiling of a multidrug-resistant K. pneumoniae ST258 strain during infection of G. mellonella, to determine if this model is suitable for large-scale virulence factor discovery in this pathogen. Our results demonstrated a dominant role for surface polysaccharides in infection, with contributions from siderophores, cell envelope proteins, purine biosynthesis genes and additional genes of unknown function. Comparison with a hypervirulent strain, ATCC 43816, revealed substantial overlap in important infection-related genes, as well as additional putative virulence factors specific to ST258, reflecting strain-dependent fitness effects. Our analysis also identified a role for the metalloregulatory protein NfeR (YqjI) in virulence. Overall, this study offers new insight into the infection fitness landscape of K. pneumoniae, and provides a framework for using the highly flexible and easily scalable G. mellonella infection model to dissect molecular virulence mechanisms of bacterial pathogens.

SUBMITTER: Bruchmann S

PROVIDER: S-EPMC7981267 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Resistance to polymyxin antibiotics is increasing. Without new antibiotic classes, combination therapy is often required. We systematically investigated bacterial killing with polymyxin-based combinations against multidrug-resistant (including polymyxin-resistant), carbapenemase-producing Klebsiella pneumoniae Monotherapies and double- and triple-combination therapies were compared to identify the most efficacious treatment using static time-kill studies (24 h, six isolates), an in vitro pharmacokinetic/pharmacodynamic model (IVM; 48 h, two isolates), and the mouse thigh infection model (24 h, six isolates). In static time-kill studies, all monotherapies (polymyxin B, rifampin, amikacin, meropenem, or minocycline) were ineffective. Initial bacterial killing was enhanced with various polymyxin B-containing double combinations; however, substantial regrowth occurred in most cases by 24 h. Most polymyxin B-containing triple combinations provided greater and more sustained killing than double combinations. Standard dosage regimens of polymyxin B (2.5 mg/kg of body weight/day), rifampin (600 mg every 12 h), and amikacin (7.5 mg/kg every 12 h) were simulated in the IVM. Against isolate ATH 16, no viable bacteria were detected across 5 to 25 h with triple therapy, with regrowth to ∼2-log10 CFU/ml occurring at 48 h. Against isolate BD 32, rapid initial killing of ∼3.5-log10 CFU/ml at 5 h was followed by a slow decline to ∼2-log10 CFU/ml at 48 h. In infected mice, polymyxin B monotherapy (60 mg/kg/day) generally was ineffective. With triple therapy (polymyxin B at 60 mg/kg/day, rifampin at 120 mg/kg/day, and amikacin at 300 mg/kg/day), at 24 h there was an ∼1.7-log10 CFU/thigh reduction compared to the starting inoculum for all six isolates. Our results demonstrate that the polymyxin B-rifampin-amikacin combination significantly enhanced in vitro and in vivo bacterial killing, providing important information for the optimization of polymyxin-based combinations in patients.

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Identifying virulence determinants of multidrug-resistant Klebsiella pneumoniae in Galleria mellonella.

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