Capsular profiling of the Cronobacter genus and the association of specific Cronobacter sakazakii and C. malonaticus capsule types with neonatal meningitis and necrotizing enterocolitis

Background: Cronobacter sakazakii and C. malonaticus can cause serious diseases especially in infants where they are associated with rare but fatal neonatal infections such as meningitis and necrotising enterocolitis. Methods: This study used 104 whole genome sequenced strains, covering all seven species in the genus, to analyse capsule associated clusters of genes involved in the biosynthesis of the O-antigen, colanic acid, bacterial cellulose, enterobacterial common antigen (ECA), and a previously uncharacterised K-antigen. Results: Phylogeny of the gnd and galF genes flanking the O-antigen region enabled the defining of 38 subgroups which are potential serotypes. Two variants of the colanic acid synthesis gene cluster (CA1 and CA2) were found which differed with the absence of galE in CA2. Cellulose (bcs genes) were present in all species, but were absent in C. sakazakii sequence type (ST) 13 and clonal complex (CC) 100 strains. The ECA locus was found in all strains. The K-antigen capsular polysaccharide Region 1 (kpsEDCS) and Region 3 (kpsMT) genes were found in all Cronobacter strains. The highly variable Region 2 genes were assigned to 2 homology groups (K1 and K2). C. sakazakii and C. malonaticus isolates with capsular type [K2:CA2:Cell+] were associated with neonatal meningitis and necrotizing enterocolitis. Other capsular types were less associated with clinical infections. Conclusion: This study proposes a new capsular typing scheme which identifies a possible important virulence trait associated with severe neonatal infections. The various capsular polysaccharide structures warrant further investigation as they could be relevant to macrophage survival, desiccation resistance, environmental survival, and biofilm formation in the hospital environment, including neonatal enteral feeding tubes

Structure of the K141 capsular polysaccharide produced by Acinetobacter baumannii isolate KZ1106 that carries KL141 at the chromosomal K locus

The structure of the K141 type capsular polysaccharide (CPS) produced by Acinetobacter baumannii KZ1106, a clinical isolate recovered from Kazakhstan in 2016, was established by sugar analyses and one- and two-dimensional 1H and 13C NMR spectroscopy. The CPS was shown to consist of branched tetrasaccharide repeating units (K-units) with the following structure:[Formula presented] This structure was found to be consistent with the genetic content of the KL141 CPS biosynthesis gene cluster at the chromosomal K locus in the KZ1106 whole genome sequence. Assignment of the encoded enzymes allowed the first sugar of the K unit to be identified, which revealed that the β-d-GlcpNAc-(1→3)-d-GlcpNAc bond is the linkage between K-units formed by the WzyKL141 polymerase.Full Tex

Revised structure of the polysaccharide from Acinetobacter baumannii LUH5551 assigned as the K63 type capsular polysaccharide

K63 capsular polysaccharide produced by Acinetobacter baumannii isolate LUH5551 (previously designated isolate O24) was re-examined using sugar analysis, Smith degradation, and one- and two-dimensional 1H and 13C NMR spectroscopy. Though previously reported as O24 consisting of linear tetrasaccharide units that include a 7-acetamido-5-acylamino form of 8-epilegionaminic acid [8eLeg5R7Ac, acylated at C5 with (S)-3-hydroxybutanoyl or acetyl (1:1)], the elucidated structure of the K63 type capsule was found to include a derivative of 5,7-diamino-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic (legionaminic) acid, Leg5Ac7R, where R is either (S)-3-hydroxybutanoyl or an acetyl group (∼1:1 ratio). This finding is consistent with the presence of the lgaABCHIFG gene module for Leg5Ac7R biosynthesis in the KL63 gene cluster at the capsular polysaccharide (CPS) biosynthesis K locus in the LUH5551 genome. The glycosyltransferases (Gtrs) and Wzy polymerase encoded by KL63 were assigned to linkages in the linear K63 tetrasaccharide unit and linkage of the K63 units.Full Tex

The K129 capsular polysaccharide produced by Acinetobacter baumannii MAR 15–4076 has the same composition as K84 but differs in the linkage between units altering the overall branching topology

Capsular polysaccharide (CPS) is a heteroglycan that coats the cell surface of most isolates of the important Gram-negative bacterial pathogen, Acinetobacter baumannii. Strain MAR 15–4076, a clinical isolate recovered in Russia in 2015, was found to carry the KL129 sequence at the CPS biosynthesis K locus. The CPS was isolated from the strain and studied by sugar analysis, Smith degradation, one- and two-dimensional 1H and 13C NMR spectroscopy. It was composed of branched pentasaccharide units that include a →3)-α-L-Rhap-(1 → 3)-α-L-Rhap-(1 → 3)-β-D-GlcpNAc-(1→ mainchain and α-D-ManpNAc-(1 → 3)-L-Rhap side branch. Though the pentasaccharide units are identical to those that make up the K84 CPS produced by A. baumannii LUH5540, the units are linked differently via the substitution of an alternate L-Rhap residue, resulting in a difference in the overall topology of the CPS. This was due to the replacement of the Wzy polymerase gene encoded at the K locus.Full Tex