A standard protocol for the murine pneumonia model to investigate antibiotic treatment effect of MDR bacteria infections

Introduction

Preclinical in vivo PK/PDmodels playa crucial role inantimicrobial efficacy investigations. Differences in the methodology in the models used are extensive and may limit the comparability, reproducibility and translation to the clinic. To support the development of new antibiotics, the COMBINE consortium has established a standard protocol for acute murine gram-negative pneumonia and initiated the characterization of susceptible as well as multi-drug resistant isolates of K.pneumoniae and P.aeruginosa isolates.

Metods: The COMBINE protocol

Important experimental variables were discussed and the relevance confirmed through a workshop with experts in the field (ref. 1 & ref. 2) and subsequently a standard protocol was suggested (figure 1). The reproducibility of the virulence of 10 selected isolates, using the standard protocol, was evaluatied at two different laboratories, SSI and PEI. The protocol contains a few non-standardized viarables or parameters because of different insititutional practices. Differences between the SSI and PEI protocols are shown in table 1. 

Results:  Virulence and reproducibility in the COMBINE standard protocol

Virulence evaluation of bacterial isolates in the COMBINE standard protocol

Out of 33 tested isolates, 15 isolates showed a suitable virulence applying the standard protocol at Statens Serum Institut. Thev irulence was confirmed in 2-3 studies for each isolate. Virulence criteria for strain selection was defined as a minimum of 1 log10 bacterial growth in the lungs and survival of mice for ≥12 hours post inoculation. These15 isolates have been made available to thes cientific community in a biorepository at DSMZ (ref.3)

Assessment of the reproducibility of the COMBINE standard protocol

10 isolates were selected for confirmatory virulence studies at the Paul-Ehrlich-Institut. The use of the COMBINE standard protocol led to good reproducibility of virulence at both laboratories. Examples of in vivo growth curves with 4 of these isolates are shown in figure 3.

Adaptation of the standard protocol for the use with MRSA 

The standard protocol was applied to methicillin resistant Staphylococcus aureus (MRSA). Clinical isolates from the respiratory tract (SSI strain collection) were selected and tested at SSI for virulence in the standard protocol. None of the 8 isolates tested met the virulence criteria. By switching to Balb/C mice, one isolate met the virulence criteria of 1 log growth in the lungs and survival of mice for ≥12 hours post inoculation (figure 4).

Conclusion

• We confirm that the virulence of 10 reference strains is reproducible across laboratories despite a slight variation of a few non-standardized variables in the standard protocol.
• Modification of the standard protocol was used to establish an MRSA pneumonia model.

Future perspective

The robustness of the standard model is being further evaluated in PK/PD studies with reference antibiotics. The generated PK/PD data, will be made available to the community for benchmarking new small molecule antibiotics in preclinical development.

Acknowledgements

We thank the GNA NOW project and in particular Mraie Atwood, Sharon Evans and Pippa Griffin from the North Briston NHS Trust for kindly sharing clinical P. aeruginosa and K. pneumoniae isolates. Also Frederikke Rosenborg, Karen Juhl, Sandra Jensen and Tina Lundager (Statens Serum Institut) for expert technical assistance.

This work has received support from the EU/EFPIA Innovative Medicines Initiative 2 Joint Undertaking (COMBINE grant n° 853967) This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA companies’ in kind contribution. This fact sheet reflects the authors' view and neither IHI nor the European Union, EFPIA or any Associated Partners are responsible for any use that can be made of the information contained herein. 

All animal studies were ethically reviewed and carried out in accordance with European Directive 2010/63/EEC and the GSK Policy on the Care, Welfare and Treatment of Animals.

COMBINE Pneumonia Model: Additional resources

• COMBINE preclinical repository with bacterial strains found to be reproducibly virulent and fulfilling performance criteria in the COMBINE standardised pneumonia mouse model.
• Data from the COMBINE Multicenter Study to Standardize a Mouse Pneumonia Model with Pseudomonas aeruginosa and Klebsiella pneumoniae for Antibiotic Development  
• Arrazuria R, Kerscher B, Huber KE, Hoover JL, Lundberg CV, Hansen JU, Sordello S, Renard S, Aranzana-Climent V, Hughes D, Gribbon P, Friberg LE, Bekeredjian-Ding I (2022): Expert workshop summary: Advancing toward a standardized murine model to evaluate treatments for antimicrobial resistance lung infections.
  Front Microbiol 13: 988725.
• Arrazuria R, Kerscher B, Huber KE, et al. Variability of murine bacterial pneumonia models used to evaluate antimicrobial agents. Frontiers in Microbiology. 2022;13:988728.