IMPORTANCE: DNA modification plays a crucial role in bacterial regulation. Despite several examples demonstrating the role of methyltransferase (MTase) enzymes in bacterial virulence, investigation of this phenomenon on a whole-genome scale has remained elusive until now. Here we used single-molecule real-time (SMRT) sequencing to determine the first complete methylome of a strain from the multidrug-resistant E. coli sequence type 131 (ST131) lineage. By interrogating the methylome computationally and with further SMRT sequencing of isogenic mutants representing previously uncharacterized MTase genes, we defined the target sequences of three novel ST131-specific MTases and determined the genomic distribution of all MTase target sequences. Using a large collection of 95 previously sequenced ST131 genomes, we identified mobile genetic elements as a major factor driving diversity in DNA methylation patterns. Overall, our analysis highlights the potential for DNA methylation to dramatically influence gene regulation at the transcriptional level within a well-defined E. coli clone.
Methods: A total of 11 isolates from respiratory cultures in intensive care unit of a 24 bed tertiary hospital obtained over a one months period and one isolate obtained from the nebuliser during environmental screening were investigated. The bacteria were identified by Phoenix 100 system. The clonal relatedness was evaluated by PFGE and semi-automated repetitive sequence-based PCR. Genotyping tests were repeated for 10 serial subcultures.
Results: PFGE and DiversiLab yielded 10 genotypic profiles for 12 isolates. Four to eight different genotypes were observed from 10 subcultures of the same isolate.
Conclusion: We conclude that, high genetic diversity and supposed multiclonal appearance of the outbreak isolates may be due to changing profiles during subcultures most probably depending on hypermutation.
MATERIALS AND METHODS: A prospective clinical study was performed on all patients undergoing ureterorenoscopy and lithotripsy for ureteral stones with obstruction between December 1, 2000 and January 31, 2002. We obtained MSU, renal pelvic urine and fragmented stones for culture and sensitivity. An analysis of the data was performed to assess statistical association.
RESULTS: A total of 73 patients who fulfilled the criteria were recruited. Of these patients 25 (34.3%) had positive stone culture, 43 (58.9%) had positive pelvic urine and 21 (28.8%) patients had positive MSU C&S. Stone and pelvic C&S were positive simultaneously in 17 (23.3%) cases, MSU and stone C&S were positive in 8 (10.9%) cases, whereas pelvic and MSU C&S were positive in 13 (16.4%) cases (p = 0.03). MSU C&S had a sensitivity of 30.2% and specificity of 73% to detect pelvic urine C&S positivity. MSU C&S had a low positive predictive value and negative predictive value (NPV) in relation to infected pelvic urine (positive predictive value = 0.62, NPV = 0.42). Pelvic urine C&S had a NPV of 0.73 in detecting noninfected stones.
CONCLUSIONS: The results of this study suggest that in obstructive uropathy secondary to a stone MSU C&S is a poor predictor of infected urine proximal to the obstruction and infected stones.