Aminoglycoside resistance genes in early members of the Acinetobacter baumannii ST78A (SMAL, Italian clone) reside in an IS26-bounded island in the chromosome.

BACKGROUND: The Acinetobacter baumannii isolate called SMAL, previously used to determine the structures of capsular polysaccharide and lipooligosaccharide, was recovered in Pavia, Italy in 2002 among the collection of aminoglycoside-resistant isolates designated as SMAL type. This type was later called the Italian clone, then ST78. ST78 isolates are now widely distributed. OBJECTIVES: To establish the resistance gene complement and the location and structure of acquired resistance regions in early members of the Italian/ST78 clone. METHODS: The draft genome of SMAL2002 was assembled from Illumina MiSeq reads. Contigs containing resistance genes were joined and located in the chromosome using PCR with custom primers. The resistance profile was determined using disc diffusion. RESULTS: SMAL2002 is an ST78A isolate and includes three aminoglycoside resistance genes, aadB (gentamicin, kanamycin, tobramycin) aphA1 (kanamycin, neomycin) and aac(6')-Ian (amikacin, kanamycin, tobramycin). The aadB gene cassette is incorporated at a secondary site in a relative of the aphA1-containing, IS26-bounded pseudo-compound transposon, PTn6020. The aac(6')-Ian gene is in an adjacent IS26-bounded structure that includes sul2 (sulphonamide) and floR (florfenicol) resistance genes. The two pseudo-compound transposons overlap and are in the chromosomal hutU gene flanked by an 8 bp target site duplication. Although aac(6')-Ian was not noticed previously, the same genes and structures were found in several available draft genomes of early ST78A isolates. CONCLUSIONS: This study highlights the importance of correlating resistance profiles with resistance gene content. The location of acquired resistance genes in the SMAL2002 chromosome represents the original location in the ST78A lineage of ST78.

Updated Analysis of the Surface Carbohydrate Gene Clusters in a Diverse Panel of Acinetobacter baumannii Isolates.

To enhance the utility of the genetically diverse panel of Acinetobacter baumannii isolates reported recently by Galac and coworkers (M. R. Galac, E. Snesrud, F. Lebreton, J. Stam, et al., Antimicrob Agents Chemother 64:e00840-20, 2020, https://doi.org/10.1128/AAC.00840-20) and to identify the novel KL and OCL, all of the gene clusters that direct the biosynthesis of capsular polysaccharide and of the outer core of lipooligosaccharide, respectively, were reexamined. The nine KL and one OCL previously recorded as novel were identified, and nine further novel KL and two OCL were found.

Acinetobacter baumannii K106 and K112: Two Structurally and Genetically Related 6-Deoxy-l-talose-Containing Capsular Polysaccharides.

Whole genome sequences of two Acinetobacter baumannii clinical isolates, 48-1789 and MAR24, revealed that they carry the KL106 and KL112 capsular polysaccharide (CPS) biosynthesis gene clusters, respectively, at the chromosomal K locus. The KL106 and KL112 gene clusters are related to the previously described KL11 and KL83 gene clusters, sharing genes for the synthesis of l-rhamnose (l-Rhap) and 6-deoxy-l-talose (l-6dTalp). CPS material isolated from 48-1789 and MAR24 was studied by sugar analysis and Smith degradation along with one- and two-dimensional 1H and 13C NMR spectroscopy. The structures of K106 and K112 oligosaccharide repeats (K units) l-6dTalp-(1→3)-D-GlcpNAc tetrasaccharide fragment share the responsible genes in the respective gene clusters. The K106 and K83 CPSs also have the same linkage between K units. The KL112 cluster includes an additional glycosyltransferase gene, Gtr183, and the K112 unit includes α l-Rhap side chain that is not found in the K106 structure. K112 further differs in the linkage between K units formed by the Wzy polymerase, and a different wzy gene is found in KL112. However, though both KL106 and KL112 share the atr8 acetyltransferase gene with KL83, only K83 is acetylated.

Acinetobacter baumannii K20 and K21 capsular polysaccharide structures establish roles for UDP-glucose dehydrogenase Ugd2, pyruvyl transferase Ptr2 and two glycosyltransferases.

Infections caused by Acinetobacter baumannii isolates from the major global clones, GC1 and GC2, are difficult to treat with antibiotics, and phage therapy, which requires extensive knowledge of the variation in the surface polysaccharides, is an option under consideration. The gene clusters directing the synthesis of capsular polysaccharide (CPS) in A. baumannii GC1 isolate A388 and GC2 isolate G21 differ by a single glycosyltransferase (gtr) gene. They include genes encoding a novel UDP-glucose dehydrogenase (Ugd2) and a putative pyruvyl transferase (Ptr2). The composition and structures of the linear K20 and K21 tetrasaccharide repeats (K units) of the CPSs isolated from A338 and G21, respectively, were established by sugar analyses and Smith degradation along with 1D and 2D 1H and 13C NMR spectroscopy. The K20 and K21 CPSs are the first known to include GlcpA produced by Ugd2 and d-galactose with an (R)-configured 4,6-pyruvic acid acetal added by Prt2. The first sugar in the tetrasaccharide K units is 2-acetamido-4-amino-2,4,6-trideoxy-d-glucose (d-QuipNAc4N) that carries a 4-N-[(S)-3-hydroxybutanoyl] group in some K units and a 4-N-acetyl group in the others. Accordingly, K unit polymerases WzyK20 and WzyK21 form a ß-d-QuipNAc4NR-(1→2)-d-Galp bond. The K20 and K21 units differ only in the configuration of the glycosidic linkages of d-GlcpNAc allowing the unique inverting glycosyltransferases Gtr43 and the retaining glycosyltransferase Gtr45 to be assigned to the formation of the ß-d-GlcpNAc-(1→4)-d-GlcpA and α-d-GlcpNAc-(1→4)-d-GlcpA linkages, respectively.

Acinetobacter baumannii isolate BAL_212 from Vietnam produces the K57 capsular polysaccharide containing a rarely occurring amino sugar N-acetylviosamine.

The structures of capsular polysaccharides (CPSs) produced by different Acinetobacter baumannii strains have proven to be invaluable in confirming the role of specific genes in the synthesis of rare sugars through the correlation of genetic content at the CPS biosynthesis locus with sugars found in corresponding CPS structures. A module of four genes (rmlA, rmlB, vioA and vioB) was identified in the KL57 capsule biosynthesis gene cluster of A. baumannii isolate BAL_212 from Vietnam. These genes were predicted to direct the synthesis of 4-acetamido-4,6-dideoxy-d-glucose (N-acetylviosamine, d-Qui4NAc) and the K57 CPS was found to contain this monosaccharide. The K57 structure was determined and, in addition to d-Qui4NAc, included three N-acetylgalactosamine residues in the main chain, with a single glucose side branch. The KL57 gene cluster has not been found in any other A. baumannii genomes, but the rmlA-rmlB-vioA-vioB module is present in the KL119 gene cluster that would likely produce a d-Qui4NAc-containing CPS.

Genetics of biosynthesis and structure of the K53 capsular polysaccharide of Acinetobacter baumannii D23 made up of a disaccharide K unit.

The KL53 capsular polysaccharide (CPS) gene cluster of Acinetobacter baumannii D23 was sequenced, and includes a single gtr gene encoding the glycosyltransferase Gtr2, and the itrA1 gene for ItrA1 that is known to initiate CPS biosynthesis with d-QuiNAc4NAc. The K53 CPS was isolated and studied by one- and two-dimensional 1H and 13C nuclear magnetic resonance (NMR) spectroscopy before and after O-deacetylation. The disaccharide K unit of the CPS was established as →3)-α-d-GalpNAcA4Ac-(1→3)-ß-d-QuipNAc4NAc-(1→, where GalNAcA and QuiNAc4NAc indicate 2-acetamido-2-deoxygalacturonic acid and 2,4-diacetamido-2,4,6-trideoxyglucose, respectively. This established the linkage formed by Gtr2. The degree of 4-O-acetylation of d-GalNAcA by Atr18, encoded at the KL53 locus, is ~55 %.

The KL24 gene cluster and a genomic island encoding a Wzy polymerase contribute genes needed for synthesis of the K24 capsular polysaccharide by the multiply antibiotic resistant Acinetobacter baumannii isolate RCH51.

The whole-genome sequence of the multiply antibiotic resistant Acinetobacter baumannii isolate RCH51 belonging to sequence type ST103 (Institut Pasteur scheme) revealed that the set of genes at the capsule locus, KL24, includes four genes predicted to direct the synthesis of 3-acetamido-3,6-dideoxy-d-galactose (d-Fuc3NAc), and this sugar was found in the capsular polysaccharide (CPS). One of these genes, fdtE, encodes a novel bifunctional protein with an N-terminal FdtA 3,4-ketoisomerase domain and a C-terminal acetyltransferase domain. KL24 lacks a gene encoding a Wzy polymerase to link the oligosaccharide K units to form the CPS found associated with isolate RCH51, and a wzy gene was found in a small genomic island (GI) near the cpn60 gene. This GI is in precisely the same location as another GI carrying wzy and atr genes recently found in several A. baumannii isolates, but it does not otherwise resemble it. The CPS isolated from RCH51, studied by sugar analysis and 1D and 2D 1H and 13C NMR spectroscopy, revealed that the K unit has a branched pentasaccharide structure made up of Gal, GalNAc and GlcNAc residues with d-Fuc3NAc as a side branch, and the K units are linked via a ß-d-GlcpNAc-(1→3)-ß-d-Galp linkage formed by the Wzy encoded by the GI. The functions of the glycosyltransferases encoded by KL24 were assigned to formation of specific bonds. A correspondence between the order of the genes in KL24 and other KL and the order of the linkages they form was noted, and this may be useful in future predictions of glycosyltransferase specificities.

Rapid customised operon assembly by yeast recombinational cloning.

We have developed a system called the Operon Assembly Protocol (OAP), which takes advantage of the homologous recombination DNA repair pathway in Saccharomyces cerevisiae to assemble full-length operons from a series of overlapping PCR products into a specially engineered yeast-Escherichia coli shuttle vector. This flexible, streamlined system can be used to assemble several operon clones simultaneously, and each clone can be expressed in the same E. coli tester strain to facilitate direct functional comparisons. We demonstrated the utility of the OAP by assembling and expressing a series of E. coli O1A O-antigen gene cluster clones containing various gene deletions or replacements. We then used these constructs to assess the substrate preferences of several Wzx flippases, which are responsible for translocation of oligosaccharide repeat units (O units) across the inner membrane during O-antigen biosynthesis. We were able to identify several O unit structural features that appear to be important determinants of Wzx substrate preference. The OAP system should be broadly applicable for the genetic manipulation of any bacterial operon and can be modified for use in other host species. It could also have potential uses in fields such as glycoengineering.

Acinetobacter baumannii K27 and K44 capsular polysaccharides have the same K unit but different structures due to the presence of distinct wzy genes in otherwise closely related K gene clusters.

Capsular polysaccharides (CPSs), from Acinetobacter baumannii isolates 1432, 4190 and NIPH 70, which have related gene content at the K locus, were examined, and the chemical structures established using 2D(1)H and(13)C NMR spectroscopy. The three isolates produce the same pentasaccharide repeat unit, which consists of 5-N-acetyl-7-N-[(S)-3-hydroxybutanoyl] (major) or 5,7-di-N-acetyl (minor) derivatives of 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic (legionaminic) acid (Leg5Ac7R), D-galactose, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine. However, the linkage between repeat units in NIPH 70 was different to that in 1432 and 4190, and this significantly alters the CPS structure. The KL27 gene cluster in 4190 and KL44 gene cluster in NIPH 70 are organized identically and contain lga genes for Leg5Ac7R synthesis, genes for the synthesis of the common sugars, as well as anitrA2 initiating transferase and four glycosyltransferases genes. They share high-level nucleotide sequence identity for corresponding genes, but differ in the wzy gene encoding the Wzy polymerase. The Wzy proteins, which have different lengths and share no similarity, would form the unrelated linkages in the K27 and K44 structures. The linkages formed by the four shared glycosyltransferases were predicted by comparison with gene clusters that synthesize related structures. These findings unambiguously identify the linkages formed by WzyK27 and WzyK44, and show that the presence of different wzy genes in otherwise closely related K gene clusters changes the structure of the CPS. This may affect its capacity as a protective barrier for A. baumannii.

K19 capsular polysaccharide of Acinetobacter baumannii is produced via a Wzy polymerase encoded in a small genomic island rather than the KL19 capsule gene cluster.

Polymerization of the oligosaccharides (K units) of complex capsular polysaccharides (CPSs) requires a Wzy polymerase, which is usually encoded in the gene cluster that directs K unit synthesis. Here, a gene cluster at the Acinetobacter K locus (KL) that lacks a wzy gene, KL19, was found in Acinetobacter baumannii ST111 isolates 28 and RBH2 recovered from hospitals in the Russian Federation and Australia, respectively. However, these isolates produced long-chain capsule, and a wzy gene was found in a 6.1 kb genomic island (GI) located adjacent to the cpn60 gene. The GI also includes an acetyltransferase gene, atr25, which is interrupted by an insertion sequence (IS) in RBH2. The capsule structure from both strains was →3)-α-d-GalpNAc-(1→4)-α-d-GalpNAcA-(1→3)-ß-d-QuipNAc4NAc-(1→, determined using NMR spectroscopy. Biosynthesis of the K unit was inferred to be initiated with QuiNAc4NAc, and hence the Wzy forms the ß-(1→3) linkage between QuipNAc4NAc and GalpNAc. The GalpNAc residue is 6-O-acetylated in isolate 28 only, showing that atr25 is responsible for this acetylation. The same GI with or without an IS in atr25 was found in draft genomes of other KL19 isolates, as well as ones carrying a closely related CPS gene cluster, KL39, which differs from KL19 only in a gene for an acyltransferase in the QuiNAc4NR synthesis pathway. Isolates carrying a KL1 variant with the wzy and atr genes each interrupted by an ISAba125 also have this GI. To our knowledge, this study is the first report of genes involved in capsule biosynthesis normally found at the KL located elsewhere in A. baumannii genomes.

5,7-di-N-acetyl-acinetaminic acid: A novel non-2-ulosonic acid found in the capsule of an Acinetobacter baumannii isolate.

An Acinetobacter baumannii global clone 1 (GC1) isolate was found to carry a novel capsule biosynthesis gene cluster, designated KL12. KL12 contains genes predicted to be involved in the synthesis of simple sugars, as well as ones for N-acetyl-L-fucosamine (L-FucpNAc) and N-acetyl-D-fucosamine (D-FucpNAc). It also contains a module of 10 genes, 6 of which are required for 5,7-di-N-acetyl-legionaminic acid synthesis. Analysis of the composition of the capsule revealed the presence of N-acetyl-D-galactosamine, L-FucpNAc and D-FucpNAc, confirming the role of fnlABC and fnr/gdr genes in the synthesis of L-FucpNAc and D-FucpNAc, respectively. A non-2-ulosonic acid, shown to be 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-altro-non-2-ulosonic acid, was also detected. This sugar has not previously been recovered from biological source, and was designated 5,7-di-N-acetyl-acinetaminic acid (Aci5Ac7Ac). Proteins encoded by novel genes, named aciABCD, were predicted to be involved in the conversion of 5,7-di-N-acetyl-legionaminic acid to Aci5Ac7Ac. A pathway for 5,7-di-N-acetyl-8-epilegionaminic acid biosynthesis was also proposed. In available A. baumannii genomes, genes for the synthesis of 5,7-di-N-acetyl-acinetaminic acid were only detected in two closely related capsule gene clusters, KL12 and KL13, which differ only in the wzy gene. KL12 and KL13 are carried by isolates belonging to clinically important clonal groups, GC1, GC2 and ST25. Genes for the synthesis of N-acyl derivatives of legionaminic acid were also found in 10 further A. baumannii capsule gene clusters, and three carried additional genes for production of 5,7-di-N-acetyl-8-epilegionaminic acid.

Structure of the K12 capsule containing 5,7-di-N-acetylacinetaminic acid from Acinetobacter baumannii isolate D36.

The repeat unit of the K12 capsular polysaccharide isolated from the Acinetobacter baumannii global clone 1 clinical isolate, D36, was elucidated by means of chemical and spectroscopical methods. The structure was shown to contain N-acetyl-D-galactosamine (D-GalpNAc), N-acetyl-D-fucosamine and N-acetyl-L-fucosamine linked together in the main chain, with the novel sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-altro-non-2-ulosonic acid (5,7-di-N-acetylacinetaminic acid or Aci5Ac7Ac), attached to D-GalpNAc as a side branch. This matched the sugar composition of the K12 capsule and the genetic content of the KL12 capsule gene cluster reported previously. D-FucpNAc was predicted to be the substrate for the initiating transferase, ItrB3, with the Wzy polymerase making a α-D-FucpNAc-(1 → 3)-D-GalpNAc linkage between the repeat units. The three glycosyltransferases encoded by KL12 are all retaining glycosyltransferases and were predicted to form specific linkages between the sugars in the K12 repeat unit.

Structure of the K2 capsule associated with the KL2 gene cluster of Acinetobacter baumannii.

The repeat unit structure of the K2 capsule from an extensively antibiotic-resistant Acinetobacter baumannii global clone 2 (GC2) strain was determined. The oligosaccharide contains three simple sugars, d-glucopyranose, d-galatopyranose and N-acetyl-d-galactosamine, and the complex sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid (Pse5Ac7Ac or pseudaminic acid), which has not previously been reported in any A. baumannii capsule. The strain was found to carry all the genes required for the synthesis of the sugars and construction of the K2 structure. The linkages catalyzed by the initiating transferase, three glycosyltransferases and the Wzy polymerase were also predicted. Examination of publicly available A. baumannii genome sequences revealed that the same gene cluster, KL2, often occurs in extensively antibiotic-resistant GC2 isolates and in further strain types. The gene module responsible for the synthesis of pseudaminic acid was also detected in four other K loci. A related module including genes for an acylated relative of pseudaminic acid was also found in two new KL types. A polymerase chain reaction scheme was developed to detect all modules containing genes for sugars based on pseudaminic acid and to specifically detect KL2.

A conjugative plasmid carrying the carbapenem resistance gene blaOXA-23 in AbaR4 in an extensively resistant GC1 Acinetobacter baumannii isolate.

OBJECTIVES: To locate the acquired bla(OXA-23) carbapenem resistance gene in an Australian A. baumannii global clone 1 (GC1) isolate. METHODS: The genome of the extensively antibiotic-resistant GC1 isolate A85 harbouring bla(OXA-23) in Tn2006 was sequenced using Illumina HiSeq, and the reads were used to generate a de novo assembly. PCR was used to assemble relevant contigs. Sequences were compared with ones in GenBank. Conjugation experiments were conducted. RESULTS: The sporadic GC1 isolate A85, recovered in 2003, was extensively resistant, exhibiting resistance to imipenem, meropenem and ticarcillin/clavulanate, to cephalosporins and fluoroquinolones and to the older antibiotics gentamicin, kanamycin and neomycin, sulfamethoxazole, trimethoprim and tetracycline. Genes for resistance to older antibiotics are in the chromosome, in an AbaR3 resistance island. A second copy of the ampC gene in Tn6168 confers cephalosporin resistance and the gyrA and parC genes have mutations leading to fluoroquinolone resistance. An 86 335 bp repAci6 plasmid, pA85-3, carrying bla(OXA-23) in Tn2006 in AbaR4, was shown to transfer imipenem, meropenem and ticarcillin/clavulanate resistance into a susceptible recipient. A85 also contains two small cryptic plasmids of 2.7 and 8.7 kb. A85 is sequence type ST126 (Oxford scheme) and carries a novel KL15 capsule locus and the OCL3 outer core locus. CONCLUSIONS: A85 represents a new GC1 lineage identified by the novel capsule locus but retains AbaR3 carrying genes for resistance to older antibiotics. Resistance to imipenem, meropenem and ticarcillin/clavulanate has been introduced into A85 by pA85-3, a repAci6 conjugative plasmid carrying Tn2006 in AbaR4.

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.

The Acinetobacter baumannii K239 capsular polysaccharide includes heptasaccharide units that are structurally related to K86 but joined by different linkages formed by different Wzy polymerases.

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 ß-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.

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: 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 ß-d-GlcpNAc-(1→3)-d-GlcpNAc bond is the linkage between K-units formed by the WzyKL141 polymerase.

Characterization of the carbapenem-resistant Acinetobacter baumannii clinical reference isolate BAL062 (CC2:KL58:OCL1): resistance properties and capsular polysaccharide structure.

The carbapenem-resistant Acinetobacter baumannii isolate BAL062 is a clinical reference isolate used in several recent experimental studies. It is from a ventilator-associated pneumonia (VAP) patient in an intensive care unit at the Hospital for Tropical Diseases (HTD), Ho Chi Minh City, Vietnam in 2009. Here, BAL062 was found to belong to the B sub-lineage of global clone 2 (GC2) isolates in the previously reported outbreak (2008 and 2012) of carbapenem-resistant VAP A. baumannii at the HTD. While related sub-lineage B outbreak isolates were extensively antibiotic-resistant and carry GC2-associated genomic resistance islands, AbGRI1, AbGRI2, and AbGRI3, BAL062 has lost AbGRI3 and three aminoglycoside resistance genes, armA, aacA4, and aphA1, leading to amikacin, tobramycin and kanamycin susceptibility. The location of Tn2008VAR found in the chromosome of this sub-lineage was also corrected. Like many of the outbreak isolates, BAL062 carries the KL58 gene cluster at the capsular polysaccharide (CPS) synthesis locus and an annotation key is provided. As information about K type is important for the development of novel CPS-targeting therapies, the BAL062 K58-type CPS structure was established using NMR spectroscopy. It is most closely related to K2 and K93, sharing similar configurations and linkages between K units, and contains the rare higher monosaccharide, 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-l-manno-non-2-ulosonic acid (5,7-di-N-acetyl-8-epipseudaminic acid; 8ePse5Ac7Ac), the 8-epimer of Pse5Ac7Ac (5,7-di-N-acetylpseudaminic acid). Inspection of publicly available A. baumannii genomes revealed a wide distribution of the KL58 locus in geographically diverse isolates belonging to several sequence types that were recovered over two decades from clinical, animal, and environmental sources.IMPORTANCEMany published experimental studies aimed at developing a clearer understanding of the pathogenicity of carbapenem-resistant Acinetobacter baumannii strains currently causing treatment failure due to extensive antibiotic resistance are undertaken using historic, laboratory-adapted isolates. However, it is ideal if not imperative that recent clinical isolates are used in such studies. The clinical reference isolate characterized here belongs to the dominant A. baumannii GC2 clone causing extensively resistant infections and has been used in various recent studies. The correlation of resistance profiles and resistance gene data is key to identifying genes available for gene knockout and complementation analyses, and we have mapped the antibiotic resistance genes to find candidates. Novel therapies, such as bacteriophage or monoclonal antibody therapies, currently under investigation as alternatives or adjuncts to antibiotic treatment to combat difficult-to-treat CRAb infections often exhibit specificity for specific structural epitopes of the capsular polysaccharide (CPS), the outer-most polysaccharide layer. Here, we have solved the structure of the CPS type found in BAL062 and other extensively resistant isolates. As consistent gene naming and annotation are important for locus identification and interpretation of experimental studies, we also have correlated automatic annotations to the standard gene names.

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 â†’ 3)-α-l-Rhap-(1 â†’ 3)-ß-d-GlcpNAc-(1→ mainchain and α-d-ManpNAc-(1 â†’ 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 O-specific polysaccharide structure and gene cluster of serotype O:12 of the Yersinia pseudotuberculosis complex, and the identification of a novel L-quinovose biosynthesis gene.

A major virulence factor for Yersinia pseudotuberculosis is lipopolysaccharide, including O-polysaccharide (OPS). Currently, the OPS based serotyping scheme for Y. pseudotuberculosis includes 21 known O-serotypes, with genetic and structural data available for 17 of them. The completion of the OPS structures and genetics of this species will enable the visualization of relationships between O-serotypes and allow for analysis of the evolutionary processes within the species that give rise to new serotypes. Here we present the OPS structure and gene cluster of serotype O:12, thus adding one more to the set of completed serotypes, and show that this serotype is present in both Y. pseudotuberculosis and the newly identified Y. similis species. The O:12 structure is shown to include two rares ugars: 4-C[(R)-1-hydroxyethyl]-3,6-dideoxy-D-xylo-hexose(D-yersiniose) and 6-deoxy-L-glucopyranose (L-quinovose).We have identified a novel putative guanine diphosphate(GDP)-L-fucose 4-epimerase gene and propose a pathway for the synthesis of GDP-L-quinovose, which extends the known GDP-L-fucose pathway.