# Bacteriophage inactivation to prevent carry-over in preclinical assays with mycobacteria

## TL;DR

This paper addresses the problem of phage carry-over in mycobacteria experiments and evaluates methods to inactivate phages to improve experimental accuracy.

## Contribution

The study evaluates and compares the effectiveness of different phage inactivation methods specifically for mycobacteriophages.

## Key findings

- Ferrous ammonium sulfate (FAS) effectively inactivated all three tested mycobacteriophages without harming M. tuberculosis viability.
- Phage carry-over is phage-dependent, with FionnbharthΔ45Δ47 showing the strongest residual activity.
- NALC-NaOH is effective for sputum decontamination and reduces phage titers in both aqueous and sputum samples.

## Abstract

Bacteriophage carry-over, the continued lytic activity of residual phages after experimental sampling, poses a significant challenge in bacteriophage research, potentially skewing experimental results, particularly in the context of preclinical studies where perceived laboratory success may not translate clinically. These inaccuracies, relevant across various bacterial species, underscore the necessity for robust mitigation strategies. This study provides an overview of strategies to mitigate phage carry-over, evaluating their performance on a combination of three mycobacteriophages with lytic activity against M. tuberculosis.

We investigated three inactivation methods, including ferrous ammonium sulfate (FAS) and citrate for in vitro applications and N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) for sputum decontamination. The citrate buffer offered limited and specific phage inactivation, but FAS was effective in inactivating all three mycobacteriophages (FionnbharthΔ45Δ47, Muddy HRMN0157-2, and Fred313cpm-1Δ33). With pH adapted to 5.0, FAS did not significantly reduce M. tuberculosis H37Rv viability after five minutes exposure. Phage carry-over was determined to be phage-dependent; FionnbharthΔ45Δ47 exhibited the most significant reduction in M. smegmatis colony forming units without phage inactivation measures. Furthermore, we showed that NALC-NaOH methods, commonly used for sputum decontamination, are highly effective in reducing phage titers in aqueous solutions and human sputum samples.

This study demonstrates the heterogenous and phage-dependent efficacy of available inactivation buffers, underscoring the critical necessity for researchers to empirically screen their specific phage isolates against chosen inactivation methods. This is crucial to ensure that observed antibacterial effects are correctly attributed to phage activity during the experimental period, rather than to uncontrolled phage carry-over, thereby enhancing the reliability and interpretability of results.