title: Rapid and Accurate Determination of Lipopolysaccharide O-Antigen Types in Klebsiella pneumoniae with a Novel PCR-Based O-Genotyping Method.
abstract: Klebsiella pneumoniae, a Gram-negative bacillus that causes life-threatening infections in both hospitalized patients and ambulatory persons, can be classified into nine lipopolysaccharide (LPS) O-antigen serotypes. The O-antigen type has important clinical and epidemiological significance. However, K. pneumoniae O serotyping is cumbersome, and the reagents are not commercially available. To overcome the limitations of conventional serotyping methods, we aimed to create a rapid and accurate PCR method for K. pneumoniae O genotyping. We sequenced the genetic determinants of LPS O antigen from serotypes O1, O2a, O2ac, O3, O4, O5, O8, O9, and O12. We established a two-step genotyping scheme, based on the two genomic regions associated with O-antigen biosynthesis. The first set of PCR primers, which detects alleles at the wzm-wzt loci of the wb gene cluster, distinguishes between O1/O2, O3, O4, O5, O8, O9, and O12. The second set of PCR primers, which detects alleles at the wbbY region, further differentiates between O1, O2a, and O2ac. We verified the specificity of O genotyping against the O-serotype reference strains. We then tested the sensitivity and specificity of O genotyping in K. pneumoniae, using the 56 K-serotype reference strains with known O serotypes determined by an inhibition enzyme-linked immunosorbent assay (iELISA). There is a very good correlation between the O genotypes and classical O serotypes. Three discrepancies were observed and resolved by nucleotide sequencing--all in favor of O genotyping. The PCR-based O genotyping, which can be easily performed in clinical and research microbiology laboratories, is a rapid and accurate method for determining the LPS O-antigen types of K. pneumoniae isolates.
authors.affiliation: Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei, Taiwan Division of Infectious Diseases, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan fangct@ntu.edu.tw.
authors.forename: Chi-Tai
authors.identifier: http://orcid.org/0000-0002-7380-1699
authors.initials: CT
authors.lastname: Fang
authors.affiliation: Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei, Taiwan.
authors.forename: Yun-Jui
authors.initials: YJ
authors.lastname: Shih
authors.affiliation: Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei, Taiwan.
authors.forename: Cheng-Man
authors.initials: CM
authors.lastname: Cheong
authors.affiliation: Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei, Taiwan.
authors.forename: Wen-Ching
authors.initials: WC
authors.lastname: Yi
mesh_terms: D000483:Alleles; D023061:Gene Order; D056426:Genetic Loci; D005838:Genotype; D006801:Humans; D007711:Klebsiella pneumoniae / Q000145:classification* / Q000235:genetics*; D058889:Molecular Typing; D005810:Multigene Family; D019081:O Antigens / Q000235:genetics*; D010802:Phylogeny; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012703:Serotyping
chemical_list: D019081:O Antigens