Rapid and Accurate Determination of Lipopolysaccharide O-Antigen Types in Klebsiella pneumoniae with a Novel PCR-Based O-Genotyping Method

<jats:title>ABSTRACT</jats:title> <jats:p> <jats:named-content content-type="genus-species">Klebsiella pneumoniae</jats:named-content> , 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, <jats:named-content content-type="genus-species">K. pneumoniae</jats:named-content> 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 <jats:named-content content-type="genus-species">K. pneumoniae</jats:named-content> 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 <jats:italic>wzm-wzt</jats:italic> loci of the <jats:italic>wb</jats:italic> gene cluster, distinguishes between O1/O2, O3, O4, O5, O8, O9, and O12. The second set of PCR primers, which detects alleles at the <jats:italic>wbbY</jats:italic> 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 <jats:named-content content-type="genus-species">K. pneumoniae</jats:named-content> , 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 <jats:named-content content-type="genus-species">K. pneumoniae</jats:named-content> isolates. </jats:p>