IS1111 insertion sequences of Coxiella burnetii: characterization and use for repetitive element PCR-based differentiation of Coxiella burnetii isolates

Abstract

Background: Coxiella burnetii contains the IS1111 transposase which is present 20 times in the Nine Mile phase I (9Mi/I) genome. A single PCR primer that binds to each IS element, and primers specific to a region approximately 500-bp upstream of each of the 20 IS1111 elements were designed. The amplified products were characterized and used to develop a repetitive element PCR genotyping method.

Results: Isolates Nine Mile phase II, Nine Mile RSA 514, Nine Mile Baca, Scottish, Ohio, Australian QD, Henzerling phase I, Henzerling phase II, M44, KAV, PAV, Q238, Q195 and WAV were tested by PCR and compared to 9Mi/I. Sequencing was used to determine the exact differences in isolates which lacked specific IS elements or produced PCR products of differing size. From this data, an algorithm was created utilizing four primer pairs that allows for differentiation of unknown isolates into five genomic groups. Additional isolates (Priscilla Q177, Idaho Q, Qiyi, Poker Cat, Q229 and Q172) and nine veterinary samples were characterized using the algorithm which resulted in their placement into three distinct genomic groups.

Conclusion: Through this study significant differences, including missing elements and sequence alterations within and near IS element coding regions, were found between the isolates tested. Further, a method for differentiation of C. burnetii isolates into one of five genomic groups was created. This algorithm may ultimately help to determine the relatedness between known and unknown isolates of C. burnetii.

Figure 1

Proposed algorithm flowchart and agarose gel photographs of corresponding PCR products. To discriminate isolates of C. burnetii using the algorithm shown, PCR is performed using the primer pairs depicted in boxes and negative PCR reactions allow for the discrimination of isolates into the genomic groups shown. Agarose gels of PCR products using primer pairs (from top to bottom) IS 9 and IS1111-1, IS 20 and IS1111-1, IS 5 and IS1111-1, and IS 14 and IS1111-1. Lanes 1, 100 bp ladder; 2, negative control; 3, 9Mi/I; 4, 9Mi/II; 5, RSA 514; 6, 9Mi/Baca; 7, Scottish; 8, Ohio; 9, Australian QD; 10, Q195; 11, Henz I; 12, Henz II; 13, M44; 14, KAV Q154; 15, PAV Q173; 16, Q238; 17, WAV.