To simulate

growth conditions in the urinary tract, K pn

To simulate

growth conditions in the urinary tract, K. pneumoniae isolates were cultured in AUM at 37° under oxygen-deprived condition. Notable difference in the growth curves was observed when K. pneumoniae clinical learn more strains were cultured anaerobically in AUM. After 27 hours incubation, five strains with the 13-kb genomic island (NK3, NK8, NK25, NK29, NK245), showed significant growth in AUM (OD600: 0.17-0.43). In contrast, little growth (OD600: 0.04-0.06) was detected for strains that do not have the 13-kb genomic island (NTUH-K2044, NK5, NK6, NK9, CG43). The turbidities (OD600) of NK8 and NTUH-K2044 at different time points during the 27-hour incubation in AUM were also measured. Note that little growth was detected in NTUH-K2044, a strain that lacks the citrate fermentation gene cluster (Figure 3), while exponential logarithmic see more phase growth was observed from 15 to 19 h in the NK8 strain that carries the 13-kb genomic island (Figure 4). Figure 3 Citrate gene

cluster permits fermentation growth in AUM for the NTUH-K2044 strain. NTUH-K2044, a strain that lacks the 13-kb genomic region; NTUH-K2044-F06C06, NTUH-K2044 transformed by a fosmid (F06C06) carrying the 13-kb genomic region responsible for citrate fermentation from NK8. Figure 4 Citrate gene cluster is necessary for fermentation growth in AUM for the NK8 strain. NK8 is a clinical strain carrying the same Dasatinib in vitro citrate fermentation genes as the sequenced reference strain, MGH 78578; NK8-Δcit, NK8 with the 13-kb genomic region disrupted at the promoter region. The initial OD600 of the inoculated AUM culture is 0.0005. To demonstrate that the citrate fermentation genes present in the 13-kb region have allowed alternative use of carbon and

energy source, MycoClean Mycoplasma Removal Kit a fosmid, F06C06, which contains the entire 13-kb region from NK8, was transformed into NTUH-K2044. As shown in Figure 3, this fosmid enabled the bacteria (NTUH-K2044-F06C06) to grow anaerobically in AUM. The logarithmic growth (from 11 to 15 h) of the fosmid-transformed clone was shifted to the left and the cells reached the stationary phase earlier than that of the NK8. This may be a result of gene copy number discrepancies between the fosmid transformants and NK8, or a result of other genetic factors specific to the NTUH-K2044 genome. Similarly, the F06C06 fosmid sequence enabled the anaerobic growth of E. coli epi300 (Epicenter Technologies, Madison, WI) transformants in AUM (data not shown). As a control, the K. pneumoniae strains NTUH-K2044, NK8, NTUH-K2044-F06C06, and NK8-Δcit were cultured anaerobically in AUM medium prepared without citrate, all four strains showed no sign of growth in 27 hours.

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