To further explore the progression of i.g. infection, we repeated the Balb/c inoculations with either EGD-e or EGD-e InlA m * tagged with a constitutive bioluminescent lux LY2606368 datasheet marker and mice were imaged for bioluminescence on each subsequent day [18]. The EGD-e InlA m * strain exhibited uniform clinical

signs of L. monocytogenes infection by day 2 [28], while these characteristics were absent from the EGD-e group even prior to sacrifice at day 3. Consistent with the clinical scores very little light was observed from the EGD-e group, while increasing light levels were obtained from the EGD-e InlA m * group on days 1 and 2, with a distinct foci evident in the abdomen in all 5 mice by day 3 (Figure 8a). Upon ex vivo imaging of the livers, a low signal was present in the gall bladder in 3 of the 5 EGD-e infected mice, whereas a much stronger signal CYT387 was found from the gall bladders of all EGD-e InlA m * (5 out of 5) infected mice, with infection across the liver also observed (Figure 8a). The EGD-e InlA m * infected gall bladders were also found to be to twice the size of the EGD-e group. Further work is necessary to determine the exact extent of gall bladder colonization

in these animals relative to hepatocyte infection. Enumeration of the livers and spleens confirmed that the EGD-e InlA m * strain produced highly reproducible i.g. infections, with the levels recovered comparable to day three i.v. Branched chain aminotransferase infections in the liver (Figure 8b). A much larger degree of variation was observed in the EGD-e group, with statistically significant differences in bacterial counts observed between the two strains (Figure 8b). The mechanism of gall bladder colonization is currently unknown [29,

30] and warrants further investigation. The EGD-e InlA m * strain is capable of establishing highly reproducible colonization of the gall bladder upon i.g. inoculation. This strain will be extremely useful in examining factors required for gastrointestinal transit and gall bladder colonization. Figure 7 Recretion of selected InlA mutations in EGD-e. A. Comparison of the invasion attributes of EGD-e and EGD-e InlA m * (Ser192Asn/Trp369Ser). Exponential phase L. monocytogenes cells (OD = 0.8) were invaded (MOI of 25:1) in triplicate for 1 h before overlaying with gentamicin. Invasion was expressed as the average cfu count per well (with standard deviation) or invasion relative to EGD-e (below graph). The graph is representative of the data from three independent experiments. B. The relative virulence of EGD-e compared against EGD-e InlA m * (tagged with pIMC3kan and pIMC3ery respectively) was accessed by competitive index after i.v. infection (1 × 104 cfu of each strain) of 15 Balb/c mice. On each subsequent day 5 mice were euthanized and spleens and livers aseptically Semaxanib clinical trial removed and enumerated.