Multiply Antibiotic-resistant Acinetobacter Baumannii Research Articles

Characterisation of an early South African multiply antibiotic-resistant global clone 1 Acinetobacter baumannii isolate

The aim of this study was to characterise an early clinical multiply antibiotic resistant Acinetobacter baumannii global clone 1 (GC1) isolate from Africa. The draft genome sequence was determined using short-read (Illumina MiSeq) sequence data and compared to other early GC1 isolates. Resistance genes and other features were identified using various bioinformatics tools. Plasmids were visualised. LUH6050, recovered in South Africa between January 1997 and January 1999, is ST1IP:ST231Ox:KL1:OCL1. Several antibiotic resistance genes (aacC1, aadA2, aphA1, catA1, sul1, and tetA(A)) reside in AbaR32. LUH6050 also includes the plasmid pRAY*, carrying the aadB gentamicin and tobramycin resistance gene, and a 29.9 kb plasmid, pLUH6050-3, carrying the msrE-mphE (macrolide resistance) and dfrA44 (trimethoprim resistance) genes and a small cryptic Rep_1 plasmid. Plasmid pLUH6050-3, a cointegrate of pA1-1 (R3-T1; RepAci1) with an R3-T33 type plasmid encoding a different Rep_3 family Rep, carries 15 pdif sites and 13 dif modules, including those that carry the mrsE-mphE and dfrA44 genes and three that include toxin-antitoxin gene pairs. The closest relative of pLUH6050-3 found in GenBank was from an unrelated 2013 Tanzanian A. baumannii isolate. The chromosome has an AbaR0-type region in comM and includes no ISAba1 copies. Similar features were found in most other sequenced lineage 1 GC1 isolates recovered prior to 2000. LUH6050 represents an early form of the GC1 lineage 1, supplementing limited information about early isolates and isolates from Africa. These data contribute to the understanding of the emergence, evolution, and dissemination of the A. baumannii GC1 clonal complex.

AbGRI4, a novel antibiotic resistance island in multiply antibiotic-resistant Acinetobacter baumannii clinical isolates.

ObjectivesTo investigate the genomic context of a novel resistance island (RI) in multiply antibiotic-resistant Acinetobacter baumannii clinical isolates and global isolates.MethodsUsing a combination of long and short reads generated from the Oxford Nanopore and Illumina platforms, contiguous chromosomes and plasmid sequences were determined. BLAST-based analysis was used to identify the RI insertion target.ResultsGenomes of four multiply antibiotic-resistant A. baumannii clinical strains, from a US hospital system, belonging to prevalent MLST ST2 (Pasteur scheme) and ST281 (Oxford scheme) clade F isolates were sequenced to completion. A class 1 integron carrying aadB (tobramycin resistance) and aadA2 (streptomycin/spectinomycin resistance) was identified. The class 1 integron was 6.8 kb, bounded by IS26 at both ends, and embedded in a new target location between an α/β-hydrolase and a reductase. Due to its novel insertion site and unique RI composition, we suggest naming this novel RI AbGRI4. Molecular analysis of global A. baumannii isolates identified multiple AbGRI4 RI variants in non-ST2 clonal lineages, including variations in the resistance gene cassettes, integron backbone and insertion breakpoints at the hydrolase gene.ConclusionsA novel RI insertion target harbouring a class 1 integron was identified in a subgroup of ST2/ST281 clinical isolates. Variants of the RI suggested evolution and horizontal transfer of the RI across clonal lineages. Long- and short-read hybrid assembly technology completely resolved the genomic context of IS-bounded RIs, which was not possible using short reads alone.

5,7-Di-N-acetyl-8-epiacinetaminic acid: A new non-2-ulosonic acid found in the K73 capsule produced by an Acinetobacter baumannii isolate from Singapore

Nonulosonic acids are found in the surface polysaccharides of many bacterial species and are often implicated in pathogenesis. Here, the structure of a novel 5,7-diacetamido-3,5,7,9-tetradeoxynon-2-ulosonic acid recovered from the capsular polysaccharide of a multiply antibiotic resistant Acinetobacter baumannii isolate was determined. The isolate carries a sugar synthesis module that differs by only a single gene from the module for the synthesis of 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-altro-non-2-ulosonic acid or 5,7-di-N-acetylacinetaminic acid, recently discovered in the capsule of another A. baumannii isolate. The new monosaccharide is the C8-epimer of acinetaminic acid (8eAci; 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-L-altro-non-2-ulosonic acid) and the C7-epimer of legionaminic acid. This monosaccharide had not previously been detected in a biological sample but had been synthesized chemically.

RCH51, a multiply antibiotic-resistant Acinetobacter baumannii ST103IP isolate, carries resistance genes in three plasmids, including a novel potentially conjugative plasmid carrying oxa235 in transposon Tn6252.

To determine the identity and context of genes conferring antibiotic resistance in a sporadic multiply antibiotic-resistant Acinetobacter baumannii recovered at Royal Children's Hospital, Brisbane. The antibiotic resistance phenotype for 23 antibiotics was determined using disc diffusion or MIC determination. The whole-genome sequence of RCH51 was determined using the Illumina HiSeq platform. Antibiotic resistance determinants were identified using ResFinder. Plasmids were recovered by transformation. Isolate RCH51 belongs to the uncommon STs ST103 IP (7-3-2-1-7-1-4) and ST514 OX (1-52-29-28-18-114-7). It was found to be resistant to sulfamethoxazole, tetracycline, gentamicin, tobramycin and kanamycin and also exhibited reduced susceptibility to imipenem (MIC 2 mg/L) and meropenem (MIC 6 mg/L). RCH51 carries the oxa235 , sul2 , floR , aadB and tet39 resistance genes, all located on plasmids. The largest of the three plasmids, pRCH51-3, is 52 789 bp and carries oxa235 in the ISAba1-bounded transposon Tn 6252 , as well as sul2 and floR . pRCH51-3 represents a new A. baumannii plasmid family that is potentially conjugative as it contains several genes predicted to encode transfer functions. However, conjugation of pRCH51-3 was not detected. The aadB and tet39 resistance genes were each found in small plasmids identical to the known plasmids pRAY*-v1 and pRCH52-1, respectively. The resistance gene complement of RCH51 was found in three plasmids. pRCH51-3, which carries the oxa235 , sul2 and floR resistance genes, represents a new, potentially conjugative A. baumannii plasmid type.

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 β-d-GlcpNAc-(1→3)-β-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.

Structure of repeating unit of the capsular polysaccharide from Acinetobacter baumannii D78 and assignment of the K4 gene cluster

The structure of the K4 capsular polysaccharide (CPS) from a multiply antibiotic resistant Acinetobacter baumannii isolate D78 was elucidated by chemical and spectroscopical analysis. The K4 repeating unit is composed entirely by aminosugars and the trisaccharide repeating unit of the backbone polysaccharide is branched with a terminal N-acetyl-galactosamine capped with pyruvate as cyclic acetal. Each residue is in the pyranose form, α configured at the anomeric center, and has the D absolute configuration. The genes responsible for the synthesis of the K4 repeat unit and its polymerization and export are found in the capsule locus KL4 present in D78. The pyruvate acetal addition to galactosamine is formed by Ptr1, a novel pyruvate transferase, encoded at this locus.

Genomic resistance island AGI1 carrying a complex class 1 integron in a multiply antibiotic-resistant ST25 Acinetobacter baumannii isolate.

The objective of this study was to locate the antibiotic resistance determinants in the multiply antibiotic-resistant Acinetobacter baumannii isolate D4. The genome was sequenced using Illumina HiSeq and assembled de novo using Velvet. PCR was used to link the relevant contigs and fill the gaps. Sequences were compared with ones in GenBank and annotated. A sporadic A. baumannii isolate D4, recovered in Sydney in 2006 from a wound, was multiply antibiotic resistant. D4 is ST25 (Institut Pasteur scheme) and exhibited resistance to third-generation cephalosporins and reduced susceptibility to ciprofloxacin, as well as resistance to aminoglycosides (gentamicin, kanamycin, neomycin and tobramycin) and further older antibiotics, nalidixic acid, sulfamethoxazole, streptomycin, spectinomycin and trimethoprim. The gyrA gene has a mutation consistent with nalidixic acid resistance. The bla PER conferring cephalosporin resistance, together with the aadB, aadA13/2, aadA2, strAB and sul1 resistance genes, are located within a 29 173 bp complex class 1 integron that includes three copies of intI1, three cassette arrays and two copies of the 3'-conserved segment. This integron is adjacent to the resG gene of an integrative genomic resistance island, AGI1 (Acinetobacter genomic island 1), with a backbone related to that of islands in the SGI1, SGI2 and PGI1 families. AGI1 is located at the 3'-end of the chromosomal trmE (formerly thdF) gene. AGI1 represents a new lineage of genomic resistance islands that belongs in the same broad group as members of the SGI1, SGI2 and PGI1 families. Genes conferring resistance to cephalosporins, aminoglycosides and sulphonamides are located in a complex class 1 integron within AGI1.

Antibiotic resistance islands in A320 (RUH134), the reference strain for Acinetobacter baumannii global clone 2

To determine the resistance genes present and the structure of resistance islands in the multiply antibiotic-resistant Acinetobacter baumannii reference strain for global clone 2, RUH134 or A320. PCR was used to detect antibiotic resistance genes and insertion sequences, and establish linkage between genes. Structures of the resistance islands were determined by PCR mapping and DNA sequencing. Bioinformatic analysis identified features. A320 carried the strA and strB (streptomycin resistance) genes and the tet(B) tetracycline resistance determinant in a genomic island, Tn6166, located in the chromosomal comM gene. At the left-hand end, Tn6166 carried Tn6022Δ1, a derivative of Tn6022 with a 2.85 kb deletion that removed the tniD gene and part of tniB and tniE. Next to Tn6022Δ1, Tn6166 carried antibiotic resistance genes in the configuration tetA(B)-tetR(B)-CR2-strB-strA and this arrangement was followed by part of the right-hand end of a transposon related to Tn6022 (Tn6021 and Tn6019). The tet(B) determinant is derived from Tn10, but is now located adjacent to the small mobile element CR2. The aacC1 (gentamicin resistance), aadA1 (streptomycin and spectinomycin resistance) and sul1 (sulphonamide resistance) genes were in a class 1 integron, the aphA1 (kanamycin and neomycin resistance), catA1 (chloramphenicol resistance) and bla(TEM) (ampicillin resistance) genes were also detected. Transposons that target a specific position in comM play an important role in the import of antibiotic resistance genes into members of both of the globally disseminated A. baumannii clones. The organization of the A320/RUH134 island differs from the AbaR3 type.

AbaR4 replaces AbaR3 in a carbapenem-resistant Acinetobacter baumannii isolate belonging to global clone 1 from an Australian hospital

To explore the diversity of genomic resistance islands in multiply antibiotic-resistant Acinetobacter baumannii isolates in global clone 1 (GC1) from Australian hospitals. PCR was used to characterize isolates, detect antibiotic resistance genes and insertion sequences and screen for genomic resistance islands. Structures of genomic islands were determined by PCR mapping and sequencing. Multilocus sequence typing was performed using the Oxford scheme. Eleven isolates that belong to GC1 were found among 90 A. baumannii isolated between 2001 and 2010 at Australian hospitals, and 5 were carbapenem resistant. Ten isolates had the features characteristic of AbaR3 and related islands, but one carbapenem-resistant isolate did not. Instead, D36 carried the bla(OXA-23) gene in transposon Tn2006, with Tn2006 in AbaR4, and AbaR4 in the chromosomal comM gene, replacing the AbaR3-type island usually associated with multiply antibiotic-resistant GC1 isolates. D36 was resistant to gentamicin, kanamycin and tobramycin due to the aadB gene cassette in the context found in plasmid pRAY and to sulfamethoxazole due to the sul2 gene. D36 was of a rare sequence type (ST), ST247. Bioinformatic analysis identified five potential transposition genes in the AbaR backbone transposons. Substantial diversity was observed among the GC1 isolates. This is the first report of AbaR4 replacing the AbaR3-type island seen in most GC1 isolates, and D36 represents a distinct new GC1 lineage. The AbaRs are members of a large, previously undocumented family of transposons that target comM.

Aminoglycoside resistance in multiply antibiotic-resistant Acinetobacter baumannii belonging to global clone 2 from Australian hospitals

To examine the distribution and context of aminoglycoside resistance genes in multiply antibiotic-resistant Acinetobacter baumannii isolates from Australia that are members of the global clone 2 and carry the bla(OXA-23) gene conferring resistance to carbapenems. Sixty-one multiply antibiotic-resistant A. baumannii strains isolated between 2000 and 2010 at six Australian hospitals that belonged to global clone 2 and carried the bla(OXA-23) gene were studied. Various molecular techniques were used to determine their relatedness and to detect antibiotic resistance genes and insertion sequences. Structures surrounding the aminoglycoside resistance genes were sequenced. The isolates all shared several antibiotic resistance genes, including the sul2 sulphonamide resistance gene, but varied in their pattern of resistance to aminoglycosides. The aminoglycoside resistance profiles of isolates were accounted for by four resistance genes-aadB, aacC1, aphA1b and aphA6-in various combinations. The aadB gene cassette was located at a secondary site on a 6 kb plasmid similar to pRAY. The aphA6 gene was in a transposon, TnaphA6, bounded by directly oriented copies of ISAba125. The aacC1 gene cassette in a class 1 integron and Tn6020 carrying aphA1b were always present together, but were not linked. Imipenem-resistant global clone 2 A. baumannii isolates containing bla(OXA-23) have been present in Australian hospitals for at least 10 years. Variation in this global clone 2 type has occurred with the introduction of various aminoglycoside resistance genes carried on a small plasmid or within transposons.

Evolution of AbaR-type genomic resistance islands in multiply antibiotic-resistant Acinetobacter baumannii

To determine if members of the European clonal lineages are present amongst multiply antibiotic-resistant Acinetobacter baumannii isolates from Australia. To search for AbaR-type genomic antibiotic resistance islands and determine the genetic organization of any AbaR detected. Two groups of multiply antibiotic-resistant A. baumannii strains isolated between 2002 and 2007 at three Sydney hospitals were studied. Various molecular techniques were used to determine whether they belong to European epidemic clones and to detect and characterize the AbaR. One group of five isolates belonged to European clone I. In these, an AbaR-type island, characterized by the presence of resistance genes and Tn6018, was present. Mapping revealed two novel AbaR configurations, AbaR6 and AbaR7, with IS26-generated deletions of 29 and 36.2 kb, respectively, relative to AbaR5. The second group contained seven isolates belonging to European clone II and, in these, a different transposon was present in the same chromosomal location as the AbaR genomic resistance islands. Multiply antibiotic-resistant A. baumannii strains belonging to both European epidemic clonal lineages I and II are found in Australian hospitals, indicating that these clones are globally distributed. Amongst the isolates examined, only the clone I strains harboured an AbaR-type resistance island, and two novel types, AbaR6 and AbaR7, carrying only four antibiotic resistance genes were identified. Hence, AbaR are continuously evolving and IS26 plays a key role in this process.