|Asokan, Kokila - UNIV. OF GEORGIA - ATHENS|
|Carlson, R. - UNIV. OF GEORGIA -ATHENS|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 5, 1999
Publication Date: N/A
Interpretive Summary: Genetic methods for identifying virulent strains of Salmonella enteritidis are not yet reliable, which makes it difficult to conduct epidemiological investigations associated with increased human illness either due to egg contamination or due to acquisition from other sources. In this manuscript, we give a first indication where to look in the genome of S. enteritidis for DNA differences between strains that vary in their ability to cause disease. Naturally occurring variation in a gene that links sugars together to produce the complex molecule lipopolysaccharide (LPS) was detected by using refined techniques that measure LPS sugars by gas liquid chromatography (GLC). To obtain these results, a water-based extraction method gave significantly better LPS yields compared to older techniques that used organic solvents. Also, glucose supplementation of cultures improved extraction by aiding separation of LPS from fatty substances in the cell wall. Thus, the water-based technique was faster, cheaper, safer and more conducive to processing large numbers of samples by decreasing organic waste. Statistical analysis of results clearly indicated that virulent strains were easily identified. This research furthers efforts to understand how S. enteritidis contaminates eggs and contributes to human illness.
Technical Abstract: Twelve human and chicken isolates of Salmonella enterica serovar Enteritidis belonging to phage types 4, 8, 13a and 23 were screened for variability in lipopolysaccharide (LPS) fatty acid, core and O-chain sugar composition. Hot water extraction increased recovery of LPS from all isolates, compared to extraction by phenol:chloroform. Isolates lacking immunoreactive O-chain yielded one of two LPS structures, and yielded 2.5 mg rhamnose/100mg crude LPS if trace repeat units were detected by gel electrophoresis, or 7.5mg rhamnose if one repeat unit was linked to core. Smooth isolates, positive for immunoreactive O-chain, were also of two types; one yielding O-chain hexoses in amounts close to that of rough isolates (5.7mg rhamnose/100mg crude LPS, 3.0mg stan. dev.), whereas the other produced three times more rhamnose (16.5mg, 1.67mg stan. dev.). Thus, rough phenotypes are common among natural isolates and can be either Rfc phenotypes, with one O-unit linked to core, or Ra chemotypes, which lack nearly all O-chain, whereas smooth isolates can be subdivided into leaky or constitutive Rfc phenotypes. These findings demonstrate that gas chromatography analysis of water-extracted crude sample accurately defines LPS subtypes versus phenol:chloroform extracts. The results indicate that naturally occurring variability in Rfc function can be used to subtype isolates of serovar Enteritidis during epidemiological investigations.