Experimental animal models

The mouse peritonitis model has been previously described and studied extensively^43,44. Here, outbred female NMRI mice (BomTac: NMRI; weight 26–30 g; Taconic, Denmark) were applied throughout the study. The animals were kept in Macrolon type III cages in groups of three and allowed free access to feed and water. Experiments were initiated after an acclimatisation period of 5 days.

Inoculation was performed by intraperitoneal (i.p.) injection of 0.5 ml bacterial suspension containing 10⁶ CFU/ml and 5% (wt/vol) mucin. At various time points of infection, the mice were anaesthetized with a subcutaneous (s.c.) injection of pre-mixed Zolazepam/Tiletamin (Zoletil, Virbac, Kolding, Denmark), Xylasin (Xysol Vet., ScanVet, Fredensborg, Denmark) and Butorphanol (Torbugesic Vet inj., Orion Pharma, Copenhagen, Denmark) prior to blood collection from total cardiocentesis, performed with a 30-gauge needle via subxiphoid access. Blood was stored in EDTA covered micro tubes (Sarstedt, Nümbrecht, Germany). After euthanasia by cervical dislocation, a peritoneal lavage was performed by injecting 2.0 ml of sterile physiological saline i.p. After 1 minute of abdominal massage, the peritoneum was opened and peritoneal lavage fluid (PLF) withdrawn with a pipette. Spleen and both kidneys were surgically removed using sterile procedures and placed in Eppendorf tubes.

All specimens were immediately placed in an insulated 4 °C cooling box for transportation and kept on ice at 4 °C until application in subsequent tests. Standard bacterial quantification and DNA purification were performed within two hours; fluorescence microscopy the subsequent day. To ensure that bacterial cells in the blood and PLF specimens did not undergo any alterations in any of the growth parameters measured (ori:ter, oriC/cell or cell size) while kept on ice at a non-permissive growth temperature⁴⁵ post harvesting, we performed a confirmatory in vitro experiment where chromosome replication analyses (qPCR and fluorescence microscopy) were performed on exponentially growing cells left on ice for up to 24 hours (Supplementary Materials). The results combined confirmed that storage up to 24 hours on ice at non-permissive growth temperatures did not alter these parameters (Supplementary Fig. ^S3).

The mouse peritonitis model was repeated in a total of 6 independent experiments, where groups of three cohabitant animals were sacrificed at various combinations of time points; 2 (n = 15), 4 (n = 9), 6 (n = 6) 8 (n = 12) or 10 (n = 9) hours of infection.

Data from repeated experiments were pooled for statistical analyses.

To examine the possibility of inducing primary bacteraemia without an established infection outside of the bloodstream, we applied a direct intravenous (i.v.) septicaemia mouse model. Mice, housing, acclimatisation and feeding procedures were identical to that of the mouse peritonitis model. However, here, the mice were inoculated via i.v. injection of 0.2 ml bacterial suspension containing 10⁶ CFU/ml, without adjuvant, into the lateral tail vein. Animals were subsequently handled as above mentioned, with the extraction of blood, peritoneal wash fluid, spleen and kidneys, which were all kept at 4 °C after harvesting. The animals were sacrificed in groups of three biological replicates at 2 (n = 3), 4 (n = 3) and 8 (n = 3) hours of infection.

Mice in both experimental models were regularly observed and scored for signs of distress. Humane end points were constituted by signs of irreversible sickness. The mice would be euthanized upon presentation of any of these signs. All animal experiments were approved by the Danish Animal Experimentation Inspectorate (Licence No. 2014-15-0201-00171) and performed according to institutional guidelines.