The structure of capsular polysaccharide (CPS) classified to K166 of Acinetobacter baumannii MAR 21-5059 strain was established using component analyses as well as 1H and 13C NMR spectroscopy, including two-dimensional homonuclear 1Н,1Н COSY, TOCSY, ROESY and heteronuclear 1Н,13C HSQC, HMBC, HSQC-TOCSY and HSQC-NOESY experiments. The CPS is composed of branched tetrasaccharide K-units containing two d-GlcNAc residues, one d-Gal and rarely occurred residue, l-RhaNAc3NAc. The following structure of the repeating K-unit was established: To our knowledge the СPS structure is novel and has not been previously published. Functions of genes in the KL166 locus of A. baumannii MAR 21-5059 were assigned by a comparison with sequences in the available databases and found to be in agreement with the CPS structure.

Klebsiella pneumoniae is a significant pathogen responsible for a range of infections, including pneumonia, urinary tract infections, and sepsis, in both human and veterinary medicine. The extracellular polysaccharide produced by this bacterium forms a capsule that protects the cell from immune responses and external influences, complicating treatment. The World Health Organization has placed this pathogen into the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) of pathogens that require special attention due to their resistance to multiple drugs, and hypermucoid K. pneumoniae strains with prevalent capsular types (e.g., K1 and K2) have been extensively studied. However, rare capsular types that are less well-characterised can still pose significant challenges. In the present study, we isolated a K. pneumoniae strain with capsular type K14, which is associated with antibiotic-resistant urogenital infections in felines, from a veterinary clinic. We successfully isolated and characterised three bacteriophages that exhibit rapid adsorption to and efficient lysis of the bacterium. All three phages demonstrated strictly lytic behavior and proved effective in degrading the extracellular polysaccharide capsule of the pathogen. We have modelled the receptor-binding proteins of these phages, which exhibit depolymerising activity. These proteins were produced in recombinant form, and experimental evidence has demonstrated their capacity to cleave the polysaccharide associated with the K14 capsular type. Furthermore, the chemical structure of the resulting cleavage products was determined. All three phages have been thoroughly characterised and show potential for application in combating multidrug-resistant K. pneumoniae.

Klebsiella grimontii is a recently described pathogen (former member of the Klebsiella oxytoca phylogroup K06), which has been elevated to the rank of a new species. The capsular polysaccharide (CPS) of K. grimontii is a principal virulence factor that significantly enhances the capacity of the bacterium to induce infections. The structure of CPS from K. grimontii strain K15g determined is this study was found to represent a novel type of polysaccharide consisting of branched hexasaccharide K-units, each composed of four monosaccharides of the main chain and a disaccharide side chain containing pyruvic acid 4,6-acetal residues. Since the CPS cleavage initiates the infection of bacterial cells by a phage, we investigated the depolymerase activity of the phage Silvester structural protein and demonstrated that the recombinant depolymerase of the phage Silvester cleaved K. grimontii K15g CPS at the β-D-GlcpA-(1→2)-α-L-Rhap linkage between the repeating units. The pathway for the synthesis of CPS of this type was proposed.

Structure of the Acinetobacter baumannii strain MAR22-3311 classified to KL205 type capsular polysaccharide (CPS) was established by component analyses, Smith degradation as well as 1H and 13C NMR spectroscopy, including two-dimensional homonuclear 1Н,1Н COSY, TOCSY, ROESY and heteronuclear 1Н,13C HSQC and HMBC experiments. The K205 CPS is composed of branched hexasaccharide K-units containing three d-GalNAc residues, and one residue each of d-Quip3NAc, d-Galp and d-Glcp. The following structure of the hexasaccharide repeating unit of the CPS was established:Functions of genes in the K locus of A. baumannii MAR22-3311 (K205) were assigned by a comparison with sequences in the available databases and found to be in agreement with the CPS structure.

Structure of the Acinetobacter baumannii strain MAR22-475 classified to KL223 type capsular polysaccharide (CPS) was established by component analyses, Smith degradation as well as 1H and 13C NMR spectroscopy, including two-dimensional homonuclear 1Н,1Н COSY, TOCSY, ROESY and heteronuclear 1Н,13C HSQC and HMBC experiments. The K223 CPS is composed of branched tetrasaccharide K-units containing two d-Glсp residues, and one residue each of d-GlcpNAc and d-Galp. The following structure of the tetrasaccharide repeating unit of the CPS was established.

The K49 capsular polysaccharide (CPS) produced by the Acinetobacter baumannii ST10 carriage isolate, NL6, that carries KL49 at the CPS biosynthesis K locus, was studied by sugar analysis along with one- and two-dimensional 1H and 13C NMR spectroscopy. The CPS was found to be comprised of linear trisaccharide units that include 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic acid (5,7-di-N-acetyl-8-epilegionaminic acid; 8eLeg5Ac7Ac), l-FucpNAc and d-GlcpNAc residues. The genetic analysis identified α-d-GlcpNAc as the first sugar, revealing that the 8eLeg5Ac7Ac-(2→3)-α-l-FucpNAc-(1→3)-α-d-GlcpNAc units are linked by WzyKL49 to form an α-GlcpNAc-(1→8)-8eLeg5Ac7Ac linkage. In addition, while KL49 includes two initiating transferase genes, NL6 harbours an ISAba1 interruption in itrB2 indicating that ItrB2, the second initiating transferase that was thought to be redundant, is not needed to make the K49 CPS. The CPS composition was found to be identical to that of a previously reported undefined polysaccharide recovered from A. baumannii ST10 isolate, LAC-4, that has since been shown to carry KL49. Hence, this polysaccharide is also a CPS.

The levels of antibodies in human blood binding to the monosaccharide α-L-Rha are the highest among all anti-glycan antibodies. Moreover, anti-Rha antibodies are found in all individuals, suggesting that they are naturally occurring rather than adaptive immunoglobulins. Rhamnose is common in both bacterial (especially infectious) and plant polysaccharides, however, it remains poorly understood which rhamnose-containing epitope(s) - whether monosaccharide, oligosaccharide, or complex molecular patterns - are recognized by human antibodies. Using an affinity adsorbent, α-L-Rha-Sepharose, antibodies were isolated from human immunoglobulin preparations (IgG + IgM + IgA) and analyzed using highly representative arrays of bacterial and plant polysaccharides (about 1000 glycans, of which >240 contained Rha). Isolated anti-α-L-Rha antibodies bound to almost all polysaccharides where the rhamnose residue was located either terminally or as α1-2, 1-3 or 1-4 linked pendant substituent, but not to internal positions, regardless of whether they were bacterial O-antigens or plant polysaccharides. It was concluded that human polyclonal anti-α-L-Rha antibodies have a reasonably narrow range of epitope specificity. The recognition of a small-sized monosaccharide epitope, on one hand, and a high proportion of IgM and IgA, on the other, suggest a high degree of polyvalence in recognizing the natural targets of the antibodies studied. In vivo, anti-α-L-Rha antibodies are more likely to recognize a pattern composed of tightly packed lipopolysaccharides (or capsular polysaccharides, or plant cell walls) rather than repetitive epitopes on a single polysaccharide molecule.

Structure of the Acinetobacter baumannii MAR20-4513 (K104) capsular polysaccharide (CPS) was established using Smith degradation, 1H and 13C NMR spectroscopy, including two-dimensional homonuclear 1Н,1Н COSY, TOCSY, ROESY and heteronuclear 1Н,13C HSQC and HMBC experiments, and modeling. The K104 CPS is composed of a branched pentasaccharide K unit containing two d-Glcp residues, and one residue each of d-GalpNAc, d-GlcpNAc and d-Galp. The following structure of the pentasaccharide repeating unit of the CPS was established:Functions of genes in the K locus of A. baumannii K104 were assigned by a comparison with sequences in the available databases and found to be in agreement with the CPS structure.

Capsular polysaccharide (CPS), a heteropolymeric carbohydrate structure present on the cell surface of most isolates of the bacterial pathogen Acinetobacter baumannii, is a major virulence determinant. Here, the CPS produced by A. baumannii MRSN 31468, which carries the KL58 CPS biosynthesis locus, was studied by sugar analysis, one- and two-dimensional 1H and 13C NMR spectroscopy. The structure was found to consist of a repeating tetrasaccharide K-unit that includes glucose (D-Glcp), galactose (D-Galp), N-acetyl-galactosamine (D-GalpNAc), and 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid (5,7-di-N-acetylpseudaminic acid; Pse5Ac7Ac). The CPS has a branched repeating unit with the disaccharide →3)-β-D-Glc-(1→3)-β-D-GalNAc-(1→ as the mainchain and O-6 of the Glc unit substituted with the disaccharide β-Pse5Ac7Ac-(2→6)-α-D-Gal, and Pse5Ac7Ac is partially acetylated at O-4. The presence of Pse5Ac7Ac in the K58 structure is consistent with the presence of psaA-F genes in KL58, which are responsible for Pse5Ac7Ac synthesis. 4-O-acetylation of Pse5Ac7Ac was traced to an acetyltransferase, Atr44, which was found to be closely related to Atr29 that similarly decorates Pse5Ac7Ac with 4OAc in the K46-type CPS. Atr44 like Atr29 is encoded by a gene found in a prophage. The K58 CPS produced by MRSN 31468 did not include the 8-epimer of Pse5Ac7Ac (5,7-di-N-acetyl-8-epipseudaminic acid; 8ePse5Ac7Ac) found in the closely related CPS from BAL062 that also carries KL58. Hence, the gene(s) for conversion of Pse5Ac7Ac to 8ePse5Ac7Ac must lie elsewhere.

Capsular polysaccharide (CPS) produced by two Acinetobacter baumannii isolates carrying the KL58 locus were recently determined and found to differ due to the presence of additional CPS biosynthesis genes found in prophage. Here, we have examined the CPS produced by a third KL58-carrying A. baumannii isolate, BAL114, which was recovered in 2009 in Vietnam. CPS extracted from BAL114 was studied by sugar analysis, selective solvolysis with trifluoroacetic acid in the presence of 2-methyl-1-propanol, and Smith degradation along with one- and two-dimensional 1H and 13C NMR spectroscopy. Consistent with the genetic content of KL58, the polysaccharide was found to include tetrasaccharide K58 units consisting of a 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic (di-N-acetylpseudaminic) acid, d-Gal, d-Glc, and d-GalNAc. However, the linkage between the K58 units was different to the β-d-GalNAc-(1→3)-d-Glc linkage seen previously for other K58 forms. There were two CPS types in the ratio ∼1 : 1 with K58 units linked by either β-(1→3) or β-(1→4) linkages between the d-GalNAc and d-Gal residues.

Structure of the capsular polysaccharide (CPS) from A. baumannii MAR21-2688 strain (classified as KL111 type) was established using 1H and 13C NMR spectroscopy, including two-dimensional homonuclear 1Н,1Н COSY, TOCSY, ROESY and heteronuclear 1Н,13C HSQC and HMBC experiments and chemical methods including component analyses and Smith degradation. The following structure of the branched hexasaccharide repeating K-unit was established: Functions of genes in the K locus of A. baumannii K111 were assigned by a comparison with sequences in the available databases and found to be in agreement with the CPS structure.

Acinetobacter baumannii is a significant nosocomial pathogen characterized by the ability to produce a wide variety of capsular polysaccharides (CPSs). The structures of a K102-type CPS isolated from A. baumannii KZ-1102 and its Smith degradation product were determined by sugar analysis, 1D and 2D 1H NMR spectroscopy, and 13C NMR spectroscopy. The K102 CPS biosynthesis gene cluster (KL102) contains genes for common sugar synthesis, K unit processing, capsule export, glycosyl transfer, initiating sugar phosphate transfer, and genes that encode d-GlcpNAc/d-GalpNAc dehydrogenase and phosphoglycerol transferase. The CPS is composed of a pentasaccharide repeating unit (K unit) consisting of a tetrasaccharide backbone including one α-d-Galp, three α-d-GlcpNAc residues, and one residue of a β-d-Glcp as a side chain. The tailspike depolymerase of the specific Obolenskvirus phage Cato was found to cleave the α-d-GlcpNAc-(1→6)-α-d-GlcpNAc linkage in the K102 CPS to give the monomer and dimer of the K repeating unit, which were characterized by high-resolution electrospray ionization mass spectrometry as well as 1H and 13C NMR spectroscopy.

The whole genome sequence from Acinetobacter baumannii MAR18-2212, an isolate recovered in 2018 from a human respiratory tract specimen in Russia, was obtained and found to carry KL95 at the capsular polysaccharide (CPS) biosynthesis K locus. KL95 includes a module of four genes (rmlB-rmlA-qdtE-qdtB) predicting synthesis of 3-acetamido-3,6-dideoxy-d-glucose (d-Quip3NAc). The structure of the K95 CPS isolated from MAR18-2212 was established using 1H and 13C NMR spectroscopy, including two-dimensional 1Н,1Н COSY, 1Н,1Н TOCSY, 1Н,1Н ROESY, 1Н,13C HSQC, and 1Н,13C HMBC experiments, and was confirmed by Smith degradation. K95 is composed of branched hexasaccharide K-units containing three d-GalpNAc residues, two d-Galp residues and one residue of d-Quip3NAc. Assignment of the enzymes encoded by KL95 established the role of rmlB-rmlA-qdtE-qdtB in the synthesis of d-Quip3NAc. Five encoded glycosyltransferases and the Wzy polymerase were also assigned to the glycosidic linkages in K95.

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: 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.

Bacteria from the genus Proteus are facultative human pathogens, primarily attacking the urinary tract and wounds. A total of 85 O serogroups have been identified so far among these bacilli. P. mirabilis Bprz 86 was isolated from the fistula of a patient in Łódź, Poland. Enzyme-Linked Immunosorbent Assay (ELISA) and Western blotting studies involving the P. mirabilis Bprz 86 lipopolysaccharide (LPS) and the strain-specific rabbit antiserum indicated that the strain, which does not belong to any of the O1–O85 serogroups, shares a common epitope with Proteus O17 antigens and is identical to another clinical P. mirabilis strain, Sm 120, isolated from the urine of a patient in the area. The O-specific polysaccharide (O antigen) was obtained from P. mirabilis Bprz 86 LPS through mild acid degradation, and the six-constituent structure of its repeating unit was determined using chemical analyses and 1D and 2D 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy. It includes (R)-3-hydroxybutanoyl, which, along with fucosamine and glucose residues, forms a fragment also present in the O17 antigens. Based on the obtained serological and chemical data, the two studied P. mirabilis isolates were proposed as candidates for a new successive O serogroup in the genus Proteus, O86.

Acinetobacter baumannii is a leading cause of multidrug-resistant bacterial infections worldwide, and the capsular polysaccharide (CPS) is a major virulence determinant. A previous study of A. baumannii from intubated and asymptomatic patients admitted to the intensive care unit (ICU) at the Hospital for Tropical Diseases in Ho Chi Minh City in Vietnam revealed multiple lineages with diverse antibiotic resistance profiles and CPS biosynthesis loci. Here, we show that 48_n, an asymptomatic nasal carriage isolate belonging to ST142, is extensively antibiotic resistant and carries acquired resistance determinants accounting for the resistance profile. 48_n carries the novel KL71 CPS biosynthesis locus in the chromosome. The structure of the CPS produced by 48_n was established using 1H and 13C nuclear magnetic resonance spectroscopy, including two-dimensional 1Н,1Н COSY, 1Н,1Н TOCSY, 1Н,1Н ROESY, 1Н,13C HSQC, and 1Н,13C HMBC experiments, and confirmed by Smith degradation. Consistent with the genetic content of KL71, the K71 CPS was found to be made up of octasaccharide K units containing six l-rhamnose residues and one residue each of N-acetyl-d-glucosamine and d-glucuronic acid. K71 CPS was branched and closely related to the K74 CPS produced by BAL_309, an antibiotic susceptible ST142 isolate recovered from an intubated patient in the same ICU 7 years later. K71 and K74 differ only in the linkage between K units, and this is due to the replacement of a single gene at the K locus that codes for the Wzy polymerase. IMPORTANCE:The majority of Acinetobacter baumannii genomes sequenced and analyzed to develop an understanding of extensively drug-resistant (XDR) isolates belong to the globally disseminated CC2 clonal complex. While XDR isolates belonging to rarer lineages are often unexplored, detailed analyses could provide novel insights into the spread of resistance, as well as cell surface features such as the CPS that determine the specificity of non-antibiotic therapeutics required to treat XDR infections that resist antimicrobial chemotherapy. Here, we describe the properties of an XDR asymptomatic nasal carriage isolate recovered in Vietnam that belongs to ST142, a rarely encountered sequence type. We report the resistance profile and correlate this with detected resistance determinants. We also solve the structure of the CPS and reveal its relationship with CPS produced by other A. baumannii isolates.

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.

The K239 type capsular polysaccharide (CPS) isolated from Acinetobacter baumannii isolate MAR19-4435 was studied by sugar analysis, one- and two-dimensional 1H and 13C NMR spectroscopy. K239 consists of branched heptasaccharide repeats (K-units) comprised of five residues of l-rhamnose (l-Rhap), and one residue each of d-glucuronic acid (d-GlcpA) and N-acetyl-d-glucosamine (d-GlcpNAc). The structure of K239 is closely related to that of the A. baumannii K86 CPS type, though the two differ in the 2,3-substitution patterns on the l-Rhap residue that is involved in the linkage between K-units in the CPS polymer. This structural difference was attributed to the presence of a gtr221 glycosyltransferase gene and a wzyKL239 polymerase gene in KL239 that replaces the gtr80 and wzyKL86 genes in the KL86 CPS biosynthesis gene cluster. Comparison of the two structures established the role of a novel WzyKL239 polymerase encoded by KL239 that forms the β-d-GlcpNAc-(1→2)-l-Rhap linkage between K239 units. A. baumannii MAR19-4435 was found to be non-susceptible to infection by the APK86 bacteriophage, which encodes a depolymerase that specifically cleaves the linkage between K-units in the K86 CPS, indicating that the difference in 2,3-substitution of l-Rhap influences the susceptibility of this isolate to bacteriophage activity.