Cronobacter Support
06-03-2009, 04:27 PM
1st International Conference on Cronobacter Poster Abstract 58
Characterization of plasmids possessed by clinical strains of Cronobacter species (formerly Enterobacter sakazakii) and the development of a PCR assay for the presence of two common plasmids.
Cronobacter species, formerly Enterobacter sakazakii (Cs) are emerging food-borne pathogens that cause sepsis, meningitis, and necrotizing enterocolitis in neonates and infants. However, little is known about the pathogenicity of these organisms. Strain BAA-894 has been shown to carry two plasmids, pESA-3 and pESA-2 (molecular sizes ~131 Kb and 31 Kb, respectively). Plasmid-specific genes include an ompT protein encoded on the larger plasmid and a VirB-Type IV secretion system (T4SS) encoded on the smaller plasmid. OmpT belongs to a family of highly homologous outer membrane proteases, known as omptins, which play a role in the virulence of Salmonella, Shigella and Yersinia spp. T4SS gene clusters in Brucella spp. and Helicobacter pylori have been shown to play a role in these organisms’ ability to persist intracellularly within host cells. It is reasonable to predict that ompT and VirB would be good plasmid markers. Plasmid profile analysis of 45 clinical isolates was performed. Plasmid DNAs were extracted using a QIAprep spin Miniprep kit (Qiagen), and then separated by agarose gel electrophoresis (AGE). Results from AGE analysis showed that one strain did not possess any plasmid. However based on size, 12 different plasmid profiles were observed among the strains. A common plasmid band was observed among plasmid positive strains. To identify which plasmid(s) was being carried, we performed polymerase chain reaction (PCR) analysis with primers targeting ompT and VirB11. Forty-two strains were found to possess the ompT gene, while 30 strains were found to possess the VirB11 gene. Also, 27 strains were positive for both plasmid targets. Interestingly, no strains found to possess one or both plasmids by AGE, were PCR negative. The strain negative by AGE analysis was also negative by PCR analysis. In summary, our data suggests that most clinical strains possess both plasmids and that the presence of the plasmids can be determined by using PCR analysis. These data also suggest that some strains carry plasmids other than pESA 3 or pESA-2. The role of these plasmids in the pathogenesis of Cs is currently under investigation.
Lan Hu, V. Sathyamoorthy, J. Kim and Ben D. Tall
Centre for Food Safety & Applied Nutrition, US Food and Drug Administration, Laurel, MD 20708, USA.
Characterization of plasmids possessed by clinical strains of Cronobacter species (formerly Enterobacter sakazakii) and the development of a PCR assay for the presence of two common plasmids.
Cronobacter species, formerly Enterobacter sakazakii (Cs) are emerging food-borne pathogens that cause sepsis, meningitis, and necrotizing enterocolitis in neonates and infants. However, little is known about the pathogenicity of these organisms. Strain BAA-894 has been shown to carry two plasmids, pESA-3 and pESA-2 (molecular sizes ~131 Kb and 31 Kb, respectively). Plasmid-specific genes include an ompT protein encoded on the larger plasmid and a VirB-Type IV secretion system (T4SS) encoded on the smaller plasmid. OmpT belongs to a family of highly homologous outer membrane proteases, known as omptins, which play a role in the virulence of Salmonella, Shigella and Yersinia spp. T4SS gene clusters in Brucella spp. and Helicobacter pylori have been shown to play a role in these organisms’ ability to persist intracellularly within host cells. It is reasonable to predict that ompT and VirB would be good plasmid markers. Plasmid profile analysis of 45 clinical isolates was performed. Plasmid DNAs were extracted using a QIAprep spin Miniprep kit (Qiagen), and then separated by agarose gel electrophoresis (AGE). Results from AGE analysis showed that one strain did not possess any plasmid. However based on size, 12 different plasmid profiles were observed among the strains. A common plasmid band was observed among plasmid positive strains. To identify which plasmid(s) was being carried, we performed polymerase chain reaction (PCR) analysis with primers targeting ompT and VirB11. Forty-two strains were found to possess the ompT gene, while 30 strains were found to possess the VirB11 gene. Also, 27 strains were positive for both plasmid targets. Interestingly, no strains found to possess one or both plasmids by AGE, were PCR negative. The strain negative by AGE analysis was also negative by PCR analysis. In summary, our data suggests that most clinical strains possess both plasmids and that the presence of the plasmids can be determined by using PCR analysis. These data also suggest that some strains carry plasmids other than pESA 3 or pESA-2. The role of these plasmids in the pathogenesis of Cs is currently under investigation.
Lan Hu, V. Sathyamoorthy, J. Kim and Ben D. Tall
Centre for Food Safety & Applied Nutrition, US Food and Drug Administration, Laurel, MD 20708, USA.