Whole blood agar

A 10–15 mL volume of blood was obtained from each rat via puncture in the vena cava inferior. The MLNs of the ileocaecal area were aseptically isolated. After the isolates were ground, 100 μL of homogenized MLNs were cultured on MacConkey (Thermo Fisher Scientific, Waltham, MA), Mueller–Hinton (Thermo Fisher Scientific), and whole blood agar (Bio Merieux, Lyon, France) for 48 hours at 37 °C. BT was defined as the presence of viable organisms in the MLN culture2 (link)29 (link)30 (link). To determine whether bacteraemia was present, 3 mL of blood was drawn from the inferior vena cava and inoculated into aerobic and anaerobic Bactec culture bottles. The cultures were incubated at 35 °C, and the growth value (a measurement of CO₂ production by the bacteria) was continuously monitored for at least 7 days4 (link). For BT monitoring, 10 cirrhotic rats and four control rats were lavaged with 10⁸ RFP-marked E. coli. The small intestine, colon, heart, lung, spleen, MLNs, kidneys, and liver were collected at 2 or 6 hours after lavage. The organs were rinsed in ice-cold PBS twice, and the RFP signal was visualised using a Clairvivo OPT Plus fluorescence microscope (Shimadzu Corporation, Kyoto, Japan) at a wavelength of 583 nm.

Bacterial translocation (BT) is generally considered as the presence of viable organisms in the MLN culture (Teltschik et al. 2012 (link)). Specifically, the MLNs were separated aseptically from the ileocecal zone. After grinding the separation, the homogenized MLNs (100 μL) were put in the MacConkey (Thermo Fisher Scientific), Mueller–Hinton (Thermo Fisher Scientific), and whole blood agar (Bio Merieux, Lyon, France) at 37 °C for approximately 2 days. Ultimately, the number of bacteria per gram was quantified using BT (CFU/g).