Steven J. Nigro

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

OBJECTIVES 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Tn6167, an antibiotic resistance island in an Australian carbapenem-resistant Acinetobacter baumannii GC2, ST92 isolate

OBJECTIVES To determine the context and location of the bla(OXA-23) carbapenem-resistance gene and the structure of the resistance island in the chromosomal comM gene in a representative Australian global clone 2 (GC2) Acinetobacter baumannii isolate. METHODS Long-range PCR was used to link genes and determine the organization of the resistance island. PCR amplicons were sequenced, and bioinformatic analysis identified features. Multilocus sequence typing (MLST) was performed. RESULTS The GC2 isolate A91 is sequence type (ST) ST92 (Oxford MLST scheme). It includes a 37 kb genomic resistance island, Tn6167, in the comM gene. At one end, Tn6167 carries Tn6022Δ1 interrupted by a novel insertion sequence, ISAba17. The sul2 (sulphonamide resistance) and strA-strB (streptomycin resistance) genes and tet(B) tetracycline resistance determinant are at the other end in the configuration ISAba1-sul2-CR2Δ-tetA(B)-tetR(B)-CR2-strB-strA with part of the tni end of a Tn6022-related transposon preceding them and an orf4 end following them. Transposon Tn2006 carrying bla(OXA-23) was found in an 11 kb region located between Tn6022Δ1 and the other resistance genes. The 17.6 kb Tn6166 from the GC2 reference strain A320/RUH134 can be derived from Tn6167 via a single deletion arising adjacent to Tn6022Δ1 and causing loss of a large central segment. CONCLUSIONS The transposons found in comM in the GC2 isolates A91 and A320 differ substantially from AbaR3-type islands, found predominantly in global clone 1 (GC1) isolates, in both resistance gene content and organization. However, the A. baumannii GC1 and GC2 clones have both acquired antibiotic resistance genes via their association with transposons that target comM.

A novel family of genomic resistance islands, AbGRI2, contributing to aminoglycoside resistance in Acinetobacter baumannii isolates belonging to global clone 2

OBJECTIVES To determine the context and location of antibiotic resistance genes in carbapenem- and aminoglycoside-resistant Acinetobacter baumannii global clone 2 (GC2) isolates carrying a class 1 integron. METHODS Isolates were from Sydney hospitals. Resistance to antibiotics was determined by disc diffusion. BLAST searches identified relevant DNA fragments in a draft genome sequence. PCR was used to assemble fragments and map equivalent regions. RESULTS In two isolates belonging to GC2, WM99c and A91, the bla(TEM) gene, the class 1 integron carrying the aacC1-orfP-orfP-orfQ-aadA1 cassette array and sul1 gene, and the aphA1b gene in Tn6020 were each in segments flanked by IS26. These, together with a fourth IS26-flanked segment, formed a 19.5 kb genomic resistance island (GRI), designated AbGRI2-1, containing five copies of IS26. Part of this island was identical to part of the multiple antibiotic resistance region of AbaR-type islands found in global clone 1 (GC1). AbGRI2-1 has replaced a 40.9 kb segment found in the AB0057 genome. Related GRIs were identified in the same location in published GC2 genomes and appear to have arisen from AbGRI2-1 via IS26-mediated deletions. Like A91, WM99c carries ISAba1 upstream of ampC and Tn6167, an AbGRI1-type island in the chromosomal comM gene containing sul2, tet(B), strA and strB genes and bla(OXA-23) in Tn2006. In WM99c, the chromosomal gene encoding OXA-Ab is interrupted by ISAba17. CONCLUSIONS AbGRI2-1 is the largest so far of a new type of GRI designated AbGRI2 to distinguish them from the islands in comM in GC1 isolates (AbaR type) and in GC2 isolates (AbGRI1 type).

Structure and context of Acinetobacter transposons carrying the oxa23 carbapenemase gene

Theoxa23gene encoding the OXA-23 carbapenemase (and several minor variants of it) is widespread inAcinetobacter baumanniiclinical isolates and compromises treatment with carbapenem antibiotics. The gene is derived from the chromosome ofAcinetobacter radioresistenswhere it is an intrinsic gene, here designatedoxaAr InA. baumanniiand otherAcinetobacterspecies,oxa23is usually preceded by an IS, ISAba1, which supplies the strong promoter required for the gene to confer clinically relevant levels of resistance. TheoxaArgene appears to have been mobilized twice creating Tn2008and Tn2008B, both of which consist of a single ISAba1 and anA. radioresistens-derived fragment. Tn2006and Tn2009are clearly derived from Tn2008Band are each made up of Tn2008Bwith an additional segment of unknown origin and an additional ISAba1, creating a compound transposon. Tn2006, Tn2008and possibly Tn2008Bare globally disseminated, while Tn2009has as yet only been found in China. Of the four ISAba1-associated transposons, Tn2006has been most frequently observed worldwide and Tn2006in Tn6022, known as AbaR4, appears to contribute significantly to the dissemination ofoxa23 Moreover, AbaR4, Tn2006, Tn2008and Tn2009have each been found in conjugative plasmids, further facilitating their spread.

Variants of the gentamicin and tobramycin resistance plasmid pRAY are widely distributed in Acinetobacter

OBJECTIVES To determine the cause of resistance to the aminoglycosides gentamicin and tobramycin in Acinetobacter isolates and the location of the resistance genes. METHODS Australian Acinetobacter baumannii isolates were screened for resistance to aminoglycosides. PCR followed by restriction digestion of amplicons was used to detect genes and plasmids. Plasmids were isolated and examined by restriction digestion. Plasmid DNA sequences were determined and bioinformatic analysis was used to identify features. The sequence of the bla(OXA-Ab) gene and multilocus sequence typing were used to determine strain types. RESULTS Isolates that exhibited resistance to gentamicin, kanamycin and tobramycin were of diverse strain types. These isolates all carried the aadB gene cassette, and in all but one the cassette was in a 6 kb plasmid similar to pRAY. The three plasmid sequences determined revealed multiple frame-shift differences in the available pRAY sequence that altered the reading frames. In pRAY*, mobA and mobC mobilization genes were identified, but no potential replication initiation protein was found. pRAY*-v1 differed from pRAY* by 66 single-base differences, and pRAY*-v2 included two insertion sequences, ISAba22, located upstream of the aadB gene cassette, and IS18-like, within ISAba22. CONCLUSIONS The plasmid pRAY* and variants are widely distributed in Acinetobacter spp. and are the most common cause of resistance to gentamicin and tobramycin. Mobilization genes should assist in the dissemination of pRAY* and its variants.

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

OBJECTIVES 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Distribution of the blaOXA-23-containing transposons Tn2006 and Tn2008 in Australian carbapenem-resistant Acinetobacter baumannii isolates

Sir, Worldwide, resistance to carbapenems, imipenem and meropenem is widespread among Acinetobacter causing infections and most resistant isolates are also resistant to many other antibiotic classes. Several class D b-lactamases can hydrolyse carbapenems, conferring resistance. The blaOXA-23 gene was the first to be found and was originally identified in a conjugative plasmid from a clinical Acinetobacter baumannii isolate recovered in the UK in 1985 and the plasmid was shown to be transferable to other Acinetobacter species, but not to Escherichia coli. The blaOXA-23 gene is now predominant among A. baumannii isolates in many countries. – 4 The blaOXA-23 gene originated in Acinetobacter radioresistens and has been mobilized into other species by ISs. However, resistance is only observed if expression is elevated by the presence upstream of a strong promoter, typically an appropriately oriented copy of ISAba1. To date, several transposons or transposon-like structures carrying blaOXA-23 have been identified. However, only Tn2006 (Figure 1a) has been shown to move and it has been found in several locations surrounded by a 9 bp duplication of the target sequence. Less is known about Tn2008, which consists of a single copy of ISAba1 adjacent to a continuous 1612 bp segment from an A. radioresistens isolate and, in the original sequence (GenBank accession number EU594641), Tn2008 is flanked by a 9 bp duplication of the target sequence (TTAATGTTT). The blaOXA-23-containing segment in both Tn2006 and Tn2008 is A. radioresistens-derived. However, it may have been picked up twice independently as the distance between the ISAba1 and the initiation codon of blaOXA-23 is 34 bp in Tn2006 and 27 bp in Tn2008 and there are single nucleotide differences in both the ISAba1 and the blaOXA-23-containing segment (Figure 1a). We recently identified a segment related to Tn2008 in a conjugative repAci6 plasmid pABUH1 (GenBank accession number AYOH01000010), but for comparison used the incorrect shorter sequence (GenBank accession number GQ861438) of Mugnier et al. rather than the original sequence (GenBank accession number EU594641). In fact, pABUH1 carries Tn2008 in the same location and flanked by the same 9 bp duplication as in the original report. To determine whether Tn2008 is present in Australian carbapenem-resistant A. baumannii isolates, the genome sequences of .100 isolates were examined for the presence of Tn2008. All of the global clone 2 (GC2) isolates described previously carried Tn2006 in AbGRI1-2 (Tn6167), as seen for A91. Among additional isolates, three 2008 GC2 isolates from Flinders Medical Centre (08325850, 08317005 and 08315000) carried Tn2008. In addition to the resistances conferred by blaOXA-23, these isolates were resistant to third-generation cephalosporins, tetracycline, spectinomycin, streptomycin, sulfamethoxazole, tetracycline and various aminoglycosides. Tn2008 was in a chromosomal gene encoding a short-chain dehydrogenase and surrounded by a different 9 bp duplication (AAGCGACTC) (Figure 1b). The sequence has been deposited in GenBank under accession number KP780408. BLAST searches were used to determine whether Tn2008 is a discrete entity that is found in further locations. A third location in the draft genomes of GC1 isolates AB5075 (GenBank accession number JHUI01000012), IS-58 (GenBank accession number AMGH01000100) and ANC 4097 (GenBank accession number APRF01000011) was identified. In this case, the interrupted gene is in the chromosome and annotated as a ‘phage-related’ membrane protein. The duplicated bases are CAATTCAAC (Figure 1b). After this work was completed, Tn2008 was found in a fourth location inside ISAba125, again surrounded by a 9 bp duplication. A further location for Tn2008 was in plasmid pNB09A30 from Acinetobacter baylyi (GenBank accession number JF731029), but in this case an additional two bases have been carried with it (Figure 1c). Tn2008 in this location interrupted by a copy of ISAba29 is found in the draft genome of the GC1 isolate Naval-83 (GenBank accession number AMFK00000000). The sequence reported as Tn2008 by Mugnier et al. appears to represent an incompletely sequenced version of Tn2008::ISAba29 (Figure 1c). The fact that Tn2008 has been found in four (potentially five) different locations, each of which is uninterrupted in other available sequences, is consistent with repeated transpositional movement of the DNA segment originally named as Tn2008, even though it includes only one ISAba1. Examination of the outer end of the blaOXA-23-containing segment revealed a match with 10 of 11 bases at the inner end of the 16 bp inverted repeats (IRs) of ISAba1, with the right-hand end of Tn2008 located 5 bp away (Figure 1d). The fortuitous presence of this potential transposase binding site in the A. radioresistens-derived segment appears to allow Tn2008 to move.

Loss and gain of aminoglycoside resistance in global clone 2 Acinetobacter baumannii in Australia via modification of genomic resistance islands and acquisition of plasmids

OBJECTIVES The objective of this study was to examine the evolution of carbapenem-resistant global clone 2 (GC2) Acinetobacter baumannii in Australia focusing on the complement of aminoglycoside resistance genes and their location in resistance islands and plasmids. METHODS Sixty-two carbapenem-resistant GC2 A. baumannii isolates with various aminoglycoside resistance profiles and resistance gene content that were recovered over the period 1999-2010 from hospitals on the east coast of Australia were examined. PCR was used to link relevant contigs retrieved from whole genomes sequenced using Illumina HiSeq and assembled de novo using Velvet. Resistance phenotypes were extended to include additional antibiotics using a disc diffusion assay. RESULTS Sixty-one isolates were ST208 (formerly ST92; Oxford scheme) and one was ST425. All isolates included the oxa23 carbapenem resistance gene in Tn2006 located in the same position in AbGRI1-2, along with the ISAba1-sul2-CR2Δ-tetA(B)-tetA(R)-CR2-strB-strA configuration. All isolates harboured either AbGRI2-1 carrying the aacC1 (gentamicin resistance) cassette or a variant derived from it via loss of some of the island content. When aacC1 was lost, aminoglycoside resistance was sometimes regained via acquisition of aadB (gentamicin, kanamycin and tobramycin resistance) in pRAY*-v1 or TnaphA6 (amikacin, kanamycin and neomycin resistance) in a repAci6 plasmid. A small cryptic plasmid or a deletion variant of this plasmid was always present and a large cryptic plasmid was also variably present. CONCLUSIONS The extensively antibiotic-resistant GC2 isolates from Sydney, Brisbane and Canberra appear to have arisen from a single import that was introduced into Australia in, or prior to, 1999 that then evolved and spread.

Variation in the OC locus of Acinetobacter baumannii genomes predicts extensive structural diversity in the lipooligosaccharide.

Lipooligosaccharide (LOS) is a complex surface structure that is linked to many pathogenic properties of Acinetobacter baumannii. In A. baumannii, the genes responsible for the synthesis of the outer core (OC) component of the LOS are located between ilvE and aspS. The content of the OC locus is usually variable within a species, and examination of 6 complete and 227 draft A. baumannii genome sequences available in GenBank non-redundant and Whole Genome Shotgun databases revealed nine distinct new types, OCL4-OCL12, in addition to the three known ones. The twelve gene clusters fell into two distinct groups, designated Group A and Group B, based on similarities in the genes present. OCL6 (Group B) was unique in that it included genes for the synthesis of L-Rhamnosep. Genetic exchange of the different configurations between strains has occurred as some OC forms were found in several different sequence types (STs). OCL1 (Group A) was the most widely distributed being present in 18 STs, and OCL6 was found in 16 STs. Variation within clones was also observed, with more than one OC locus type found in the two globally disseminated clones, GC1 and GC2, that include the majority of multiply antibiotic resistant isolates. OCL1 was the most abundant gene cluster in both GC1 and GC2 genomes but GC1 isolates also carried OCL2, OCL3 or OCL5, and OCL3 was also present in GC2. As replacement of the OC locus in the major global clones indicates the presence of sub-lineages, a PCR typing scheme was developed to rapidly distinguish Group A and Group B types, and to distinguish the specific forms found in GC1 and GC2 isolates.

Evolution of AbGRI2-0, the Progenitor of the AbGRI2 Resistance Island in Global Clone 2 of Acinetobacter baumannii.

ABSTRACT A320, isolated in the Netherlands in 1982 and also known as RUH134, is the earliest available multiply antibiotic-resistant (MAR) Acinetobacter baumannii isolate belonging to global clone 2 (GC2) and is the reference strain for this clone. The draft genome sequence of A320 was used to investigate the original location and configuration of the IS26-bounded AbGRI2 resistance island found in current GC2 isolates. PCR mapping and sequencing were used to order contigs composing the resistance islands. A320 contains two IS26-bounded resistance islands, AbGRI2-0a and AbGRI2-0b, of 7.8 kb and 25.4 kb, respectively. Together they contain blaTEM, aacC1, aadA1, sul1, catA1, and aphA1b genes, which confer resistance to antibiotics used clinically in the 1970s, as well as an incomplete mercury resistance module. Tracking the continuity of the chromosome and the target site duplications revealed that the two resistance islands were originally together as AbGRI2-0, an island of 32.4 kb, and were subsequently separated via an IS26-mediated intramolecular inversion that reversed the orientation of 1.54 Mb of the chromosome and duplicated an IS26. A320 contains an ancestral form of AbGRI2, and the original insertion site of the AbGRI2 island was identified. Many of the AbGRI2 versions present in the completed GC2 genomes can be derived from it via the variant AbGRI2-1. IS26-mediated inversions have also played a part in forming AbGRI2-0, and, upon reversal, large regions of AbGRI2-0 are identical to parts of AbaR0, the ancestral version of the AbaR islands present in GC1 isolates. This indicates a common source.