Aerosolisation’s risk assessment of bacteriophages handled in a biological safety cabinet

B. Lapras1,4,C. Moreau1, M. Medina2,4, C. Marchand1,4, C. Merienne1,4, C. Kolenda2,4, T. Briot3,4, G. Leboucher3,4, F. Laurent2,4, F. Pirot1,4
1 Plateforme FRIPHARM, Pharmacie à usage intérieur, Groupement Hospitalier Centre - Hospices Civils de Lyon (HCL)
2 Laboratoire de bactériologie, Centre national de référence des staphylocoques – Hospices Civils de Lyon
3 Pharmacie à usage intérieur, Groupement Hospitalier Nord – Hospices Civils de Lyon
4 Consortium PHAG-ONE – PHAGinLyon

The pharmaceutical process of purification, concentration and conditioning of bacteriophage (phage) suspensions preferably involves strict aseptic conditions. These operations need to be confined – e.g., in a biological safety cabinet (BSC) – to avoid the contamination of equipment, environment and employees. However, phages are likely to generate aerosols, which can lead to cross-contaminations. The risk of phage aerosolisation therefore needs to be assessed and controlled.

In this work, we report the risk assessment of aerosol formation of phages handled in a BSC and further cross-contamination.

Materials and methods
For this study, anti-Staphylococcus aureus phages of the Myoviridae family, produced by the Hospices Civils de Lyon, were handled in a Class II BSC. For each condition tested, three Petri dishes, of 144 cm² each, filled with 25 mL of Trypto Casein Soy broth medium, were aligned diagonally in the BSC as to cover a fourth of the workbench’s surface. Containers filled with 30 mL of phage suspension - 1010 PFU/mL - were placed in the BSC as stated opposite: (i) container opened in the BSC, (ii) closed container shaken for 5 minutes then opened. A negative control – broth medium unexposed to phages – and a positive control – phage suspension diluted fifty-fold in broth medium – were added to the assay.
After 20 minutes, in order to amplify the number of phages in suspension, the broth medium was collected separately and inoculated with a strain of Staphylococcus aureus for 18 h. It was then centrifuged to separate phages and bacteria. The supernatant – containing phages – was tittered to determine the active phage concentration.

Results and discussion
Contamination of the broth medium by phages – titter of 109 PFU/mL – happened for the shaken container and the positive control. Specifically, only the broth medium of the Petri dishes placed in a 20 cm radius around the shaken container was contaminated by phages. The unshaken container and negative control were not contaminated by phages – no lysis of bacteria was observed.
The collected data suggests that the airflow tackles phage particles on the workbench’s surface of the BSC allowing the aerosol dispersion to stay localised. Moreover, under normal operating conditions, it is unlikely to shake as strongly the phage suspensions or to have prolonged opening of the phage containers. The risk of contamination by aerosolised phages therefore appears to be low.

The tested methodology did not show any crossed contamination by aerosolised phages while working in a BSC under normal operating conditions.

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