Correlation of wbiI genotype, serotype, and isolate source within species of the Burkholderia cepacia complex

Abstract

Gram-negative bacteria of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that can infect the lungs of cystic fibrosis (CF) patients and can be transmitted among these patients, causing epidemics in the CF community. Lipopolysaccharide (LPS) is an important virulence factor of many gram-negative bacteria, with the O antigen component of LPS being responsible for serotype specificity. The goal of this work was to develop a genetic method of determining the serotype of Bcc isolates based on the conserved gene wbiI. Homologues of wbiI are found in polysaccharide biosynthesis gene clusters in other bacteria. Primers to a conserved region of the Bcc wbiI gene were able to amplify by PCR a single product in 67 of 80 Bcc isolates tested. Sequencing and restriction enzyme digestion of this wbiI PCR product revealed sufficient DNA polymorphisms to distinguish and group various isolates. In five of nine instances, Bcc isolates of a single serotype had a single wbiI restriction fragment length polymorphism (RFLP) pattern, while isolates of the other four serotypes could have multiple wbiI RFLP types. Species determination of the Bcc isolates revealed no obvious correlation between wbiI RFLP type and species. There was also no apparent correlation between wbiI RFLP type and the ability of a single Bcc isolate to infect an individual with CF. However three of five Bcc outbreaks involved isolates with the same wbiI RFLP type, indicating that wbiI RFLP typing may be a useful tool to help track Bcc outbreaks.

FIG. 1.

Alignment of J2315 wbiI homologues. Numbers at the ends of each sequence represent nucleotide designations in the whole gene. The consensus sequence is shown on the next-to-last line. Underlined sequences were used to design primers Mconfor and Mconrev, shown on the bottom line. Homologues used are as follows: wbiI from J2315 (www.sanger.ac.uk), wbiI from Burkholderia pseudomallei (AF064070), wbpM from P. aeruginosa PAO1 (www.pseudomonas.com), cap5D from S. aureus (U81973), wbfY from V. cholerae (AB012957), flaA1 HP0804 from Helicobacter pylori 26695 (AE000595), cap8E from S. aureus (U73374).

FIG. 2.

PCR products from Bcc serotype O1 to O9 strains obtained with primers Mconfor and Mconrev to a conserved region of the wbiI gene. DNA size markers in base pairs are on the left. Lane 1, CIP 8235 (serotype O1); lane 2, CIP 8236 (O2); lane 3, CIP 8237 (O3); lane 4, CIP 8238 (O4); lane 5, CIP 8239 (O5); lane 6, CIP 8240 (O6); lane 7, ATCC 17759 (O7); lane 8, CDC99 (O8); lane 9, CDC86 (O9).

FIG. 3.

RFLP of wbiI PCR products from the Bcc O1 to O9 serotype strains. wbiI PCR products from Bcc O1 to O9 serotype strains were digested with BglI (top) or MboII (bottom). Four different patterns observed for each restriction enzyme digestion, labeled A to D, are shown below. Lanes 1 to 9 are as in Fig. 2, DNA size markers are shown to the left. The extra band in lane 2 is also observed in the undigested PCR product and is thus not included in pattern designation.