Comparison of an espB gene fecal polymerase chain reaction assay with bacteriologic isolation for detection of Citrobacter rodentium infection in mice.
OBJECTIVE
To develop a polymerase chain reaction (PCR) assay for specific detection of Citrobacter rodentium in fecal samples of mice and to compare this assay with bacterial isolation and identification methods.


METHODS
The target sequence of the PCR assay was the espB gene encoding a secreted virulence factor. To facilitate visual identification during primary isolation on MacConkey agar containing ampicillin, C. rodentium ATCC type strain 51459 was transformed by use of a plasmid encoding the enhanced green fluorescent protein (EGFP) and ampicillin resistance. The EGFP-C. rodentium was inoculated into Swiss Webster (SW) mice to study the time course of detection of the organism by use of fecal PCR analysis, bacterial isolation, and development of colonic hyperplasia by light microscopy. Lactose-fermenting fluorescent bacterial colonies identified during primary isolation of fecal bacteria on MacConkey-ampicillin agar were identified by use of biochemical typing.


RESULTS
Mice inoculated with EGFP-transformed C. rodentium developed colonic mucosal hyperplasia, characterized by a three-fold increase in colonic crypt height that peaked at post-inoculation day (PID) 14. The espB PCR assay detected as little as 0.3 colony-forming units of C. rodentium. The PCR assay was specific in that it did not detect the espB gene of Escherichia coli 0157. Results of in vivo studies in SW mice indicated that EGFP-C. rodentium could be detected by use of espB fecal PCR analysis in 100% of inoculated mice tested on PID 1, 3, 7, and 8, in 60% on PID 9, and in 20% on PID 10 (n = 5). Bacterial isolation from the same fecal samples detected the organism in 100% of the inoculated mice on PID 7, in 50% on PID 8, and in none on subsequent PID 9-14. The ability of the PCR assay to detect C. rodentium in fresh feces of inoculated mice was significantly better than that of bacterial isolation methods (Fisher-Irwin exact test, P < 0.01). At the time of peak colonic hyperplasia, the organism could no longer be cultivated or detected in mice by use of fecal PCR analysis.


CONCLUSIONS
The EGFP-C. rodentium was capable of inducing transmissible murine colonic hyperplasia similar to that previously reported in SW mice. The PCR assay for detection of the espB gene sequence of C. rodentium in total fecal DNA was a more sensitive diagnostic assay than was bacterial isolation.
McKeel
Rebecca
R
Center for Comparative Medicine, University of Virginia, Charlottesville, USA.
Douris
Nicholas
N
Foley
Patricia L
PL
Feldman
Sanford H
SH
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001431:Bacteriological Techniques; D016971:Citrobacter freundii / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D003106:Colon / Q000382:microbiology / Q000473:pathology; D004269:DNA, Bacterial / Q000032:analysis; D004756:Enterobacteriaceae Infections / Q000175:diagnosis / Q000662:veterinary*; D029968:Escherichia coli Proteins; D005243:Feces / Q000382:microbiology*; D005260:Female; D049452:Green Fluorescent Proteins; D006965:Hyperplasia; D007413:Intestinal Mucosa / Q000382:microbiology / Q000473:pathology; D008164:Luminescent Proteins; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013047:Specific Pathogen-Free Organisms
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; C087863:EaeB protein, E coli; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins