https://orcid.org/0000-0001-8215-916X
Abstract
To reconstruct the evolutionary history of the clinical Acinetobacter baumannii XH1056, which lacks the Oxford scheme allele gdhB.
Susceptibility testing was performed using broth microdilution and agar dilution. The whole-genome sequence of XH1056 was determined using the Illumina and Oxford Nanopore platforms. MLST was performed using the Pasteur scheme and the Oxford scheme. Antibiotic resistance genes were identified using ABRicate.
XH1056 was resistant to all antibiotics tested, apart from colistin, tigecycline and eravacycline. MLST using the Pasteur scheme assigned XH1056 to ST256. However, XH1056 could not be typed with the Oxford MLST scheme as gdhB is not present. Comparative analyses revealed that XH1056 contains a 52 933 bp region acquired from a global clone 2 (GC2) isolate, but is otherwise closely related to the ST23 A. baumannii XH858. The acquired region in XH1056 also contains a 34 932 bp resistance island that resembles AbGRI3 and contains the armA, msrE-mphE, sul1, blaPER-1, aadA1, cmlA1, aadA2, blaCARB-2 and ere(B) resistance genes. Comparison of the XH1056 chromosome to that of GC2 isolate XH859 revealed that the island in XH1056 is in the same chromosomal region as that in XH859. As this island is not in the standard AbGRI3 position, it was named AbGRI5.
XH1056 is a hybrid isolate generated by the acquisition of a chromosomal segment from a GC2 isolate that contains a resistance island in a new location—AbGRI5. As well as generating ST256, it appears likely that a single recombination event is also responsible for the acquisition of AbGRI5 and its associated antibiotic resistance genes.
Introduction
Acinetobacter baumannii is an important nosocomial pathogen that has emerged as a major concern in nosocomial settings due to its ability to rapidly acquire antibiotic resistance genes and to persist in hospital environments.1 MLST remains the primary method for initially characterizing A. baumannii isolates, even when complete genome sequences are available.2 After investigation of the population structure of A. baumannii, two 7-gene MLST schemes have been devised: the Oxford scheme introduced by Bartual et al.3 in 2005 and the Pasteur scheme introduced by Diancourt et al.4 in 2010.4 A recent comparison of the schemes, which share three loci, concluded that while the Pasteur scheme is accurate, it does not discriminate closely related isolates as well as the Oxford scheme.5 However, the Oxford scheme is beset by a number of technical issues, including the presence of two copies of the gdhB locus in 75.8% of A. baumannii genomes, which has resulted in misidentifications and the generation of incorrect STs.5 Moreover, an isolate with ISAba125 inserted in gdhB has been reported,6 highlighting the potential for this gene to be interrupted or lost, confounding typing with the Oxford MLST scheme. Here, we report the complete genome sequence of A. baumannii XH1056, which is ST256 according to the Pasteur scheme, but cannot be typed with the Oxford MLST scheme due to the absence of gdhB. We have determined that gdhB was lost due to a deletion event mediated by a copy of IS26 found at the boundary of a genomic resistance island. Close examination of this island and its context revealed that it resembles A. baumannii genomic resistance island 3 (AbGRI3) and was acquired from a global clone 2 (GC2) isolate where it had inserted at a different chromosomal position to AbGRI3. This novel resistance island position was named AbGRI5.
Materials and methods
Bacterial strain
A. baumannii XH1056 was isolated in 2009 from a sputum sample obtained from a male patient in a hospital in Hebei Province, China. The strain was cultured on Mueller–Hinton agar plates or broth (Oxoid, Hampshire, UK) at 37°C overnight. The MICs of colistin, tigecycline and eravacycline were determined using the broth microdilution method. Agar dilution was used to determine the MICs of ceftazidime, cefepime, cefoperazone/sulbactam, imipenem, meropenem, ciprofloxacin, amikacin and gentamicin. Tigecycline susceptibility data were interpreted using breakpoints for Enterobacteriaceae recommended by EUCAST,7 while susceptibility profiles for the remaining antimicrobial agents were interpreted according to Acinetobacter spp. breakpoints recommended by CLSI.8
Genomic DNA sequencing and bioinformatics analysis
Genomic DNA was extracted from XH1056 and sequenced using the Illumina HiSeq and Nanopore MinION platforms. Long-read library preparation for Nanopore sequencing was performed with a 1D sequencing kit (SQK-LSK109; Nanopore) without fragmentation. The libraries were then sequenced on a MinION device with a 1D flow cell (FLO-MIN106; Nanopore) and base-called with Guppy v2.3.5 (Nanopore). The long-read and short-read sequence data were used in a hybrid de novo assembly using Unicycler v0.4.8,9 then polished with Pilon v1.23.10 Antibiotic resistance genes were identified using the ResFinder database11 with ABRicate v0.8 (https://github.com/tseemann/abricate). MLST was performed using mlst (https://github.com/tseemann/mlst). The Nanopore long-read data were aligned to reference genomes using nglmr v0.2.7.12 The genome sequence of XH1056 was compared with XH858 (available from GenBank accession CP014528) using Mauve v2.3.1,13 as XH858 was closely related to XH1056 according to Pasteur MLST. Sequence comparisons were performed using BLAST and visualized with Easyfig v2.2.2.14
Sequence database
The full sequence of strain XH1056 has been deposited in the GenBank nucleotide database under accession number CP045645.
Results and discussion
Sequence typing XH1056
The complete, assembled genome of A. baumanniiXH1056 consists of a 4 205 854 bp chromosome (G + C content of 38.96%) and no plasmids. According to the Pasteur MLST scheme, XH1056 is ST256 (cpn60-1, fusA3, gltA10, pyrG1, recA2, rplB4, rpoB4). However, the isolate could not be typed using the Oxford scheme as the gdhB gene is absent. Interestingly, the alternative gdhB locus reported in 75.8% (553/730) of A. baumannii genomes examined in a previous study5 is also absent from XH1056.
The Pasteur scheme MLST profile of XH1056 (ST256) closely matches that of ST23 A. baumannii isolate XH858 (cpn60-1, fusA3, gltA10, pyrG1, recA4, rplB4, rpoB4), which was isolated in 2009 from a patient sputum sample in a hospital in Hangzhou, China. The MLST profiles of XH1056 and XH858 differ only at the recA allele (by two SNPs: position 90, A > G and position 135, C > T), where XH1056 carries the recA2 allele that is normally seen in isolates of A. baumannii GC2, which are typed as ST2 by the Pasteur MLST scheme.5 This suggests that XH1056 has acquired part of the GC2 chromosome via homologous recombination, similar to the acquisition of parts of the GC2 chromosome by GC1 A. baumannii.15 To determine the extent of the acquired chromosomal region, the sequences adjacent to the recA allele in XH1056, XH858 and the GC2 isolate MDR-ZJ06 (GenBank accession CP001937.2) were compared. This analysis revealed that recA2 in XH1056 is part of a 52 933 bp region that is identical to the corresponding part of MDR-ZJ06, apart from the presence of a 34 932 bp antibiotic resistance island in XH1056 (described below) that appears to replace a 16 232 bp chromosomal region found in MDR-ZJ06 (Figure 1a). The 8751 bp and 9234 bp regions on either side of the resistance island in XH1056 are 98.9% and 98.5% identical to the corresponding parts of the XH858 chromosome, respectively (Figure 1a). However, at the end of the 8751 bp fragment farthest from the resistance island, 385 bp are identical to XH858, and at the end of the 9234 bp fragment farthest from the resistance island, 40 bp are identical to XH858 (Figure 1a). These represent likely crossover sites that facilitated the homologous recombination event that led to the acquisition of the GC2 chromosomal fragment by an ST23 isolate and the generation of ST256.
AbGRI5 of A. baumannii XH1056. (a) Scaled, linear diagrams comparing chromosomal sequences of A. baumannii MDR-ZJ06, A. baumannii XH1056 and A. baumannii XH858. Insertion elements are shown as green rectangles. Housekeeping gene recA is shown as a circle with alleles distinguished by colour. Drawn to scale from GenBank accessions NC_017171.2, CP045645 and CP014528. (b) Structure of chromosomal antibiotic resistance island AbGRI5 in A. baumannii XH1056. Thicker boxes represent ISs IS26 (green), ISAba24 (grey), ISEc29 (cyan), ISEc28 (blue) and IS91 (white) or CR1 (red). Genes are shown as labelled black arrows. Drawn to scale from GenBank accession CP045645. (c) Scaled, linear diagrams comparing the sequences of A. baumannii RJ458, A. baumannii XH1056, A. baumannii MDR-ZJ06 and Tn2610. Insertion elements are shown as green arrows. Genes, including antibiotic resistance genes, are shown as labelled purple arrows. Drawn to scale from GenBank accessions KU133343, CP045645, CP001937.2 and AB207867.1. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.
Antibiotic resistance genes in XH1056
A. baumannii XH1056 was resistant to all antimicrobial agents tested, apart from colistin, tigecycline and eravacycline. Consistent with this, ResFinder identified two intrinsic chromosomal genes, blaOXA-68 and blaADC-76, and 14 antimicrobial resistance genes in the XH1056 genome, including ones that confer resistance to β-lactams (blaCARB-2, blaPER-1, blaOXA-23), aminoglycosides (armA, strAB, aadA2), tetracycline [tet(B)], sulphonamides (sul1, sul2), macrolides [ere(B), msrE, mphE] and chloramphenicol (cmlA1). The blaOXA-68 and blaADC-76 genes are not acquired genes, but instead represent the intrinsic genes for OXA-51 and AmpC β-lactamases found in A. baumannii. These genes confer only low levels of β-lactam resistance unless their expression is increased by promoters in ISs inserted upstream,16,17 and in XH1056, no ISs are found upstream of either gene.
Acquired resistance genes in XH1056 are found in three locations. The carbapenem resistance gene blaOXA-23 is located in a copy of Tn2009 flanked by the 9 bp target site duplication (TSD) AAAATATTT in an ORF for a hypothetical protein equivalent to locus ABZJ_00997 of MDR-ZJ06. Tn2009 is in the same position in ST23 strain XH858. In a second location, the strAB, sul2 and tet(B) genes are in the configuration ISAba1-sul2-CR2Δ-tetA(B)-tetR(B)-CR2-strB-strA, as in Tn6167.18 These resistance genes are part of an insertion comprised of a 14 299 bp derivative of Tn6167 (the segment from the IRl adjacent to tniCb to the IRr adjacent to orf4b) that has inserted in the ORF that corresponds to ABZJ_02949 of MDR-ZJ06 and generated the 5 bp TSD GTTGG. In XH1056 and XH858, the comM gene, which Tn6167 usually inserts into, is interrupted by a 17 828 bp element that has generated the 5 bp TSD GCGGT. The remaining resistance genes in XH1056 are found together in a resistance island that has been named AbGRI5.
AbGRI5
Most antibiotic resistance genes in XH1056 are part of a 34 932 bp IS26-bounded resistance island (Figure 1b), where both copies of IS26 are the IS26-v3 variant, which exhibits enhanced transposition activity.19 The resistance island is flanked by chromosomal sequence derived from GC2 and resembles AbGRI3, several variants of which have been described.20 However, using the complete sequence of AbGRI3 from XH1056 to query the GenBank non-redundant nucleotide database revealed that an identical island has not been sequenced previously. Furthermore, the island is not located in the chromosomal region that AbGRI3 usually occupies in GC2 (see below), so was given the name AbGRI5 as another island was recently named AbGRI4.21
The left half of AbGRI5 (Figure 1b) is identical to the corresponding part of typical AbGRI3 islands,20 comprising a partial repAciN gene that has been truncated by IS26, a copy of ISAba24 flanked by the 8 bp TSD TTATCAAG, the msrE-mphE genes, a copy of ISEc29, the aminoglycoside resistance gene armA and ISEc28. In the centre of AbGRI5 (Figure 1b) is a 6861 bp region that contains blaPER-1 and a copy of sul1 with a nonsense mutation that introduces a premature stop codon. This region is flanked by copies of CR1 (Figure 1b) and is similar to that found in RJ458 (GenBank accession KU133343.1) (Figure 1c). A single copy of CR1 is found at this position in other variants of AbGRI320 and it appears that blaPER-1 was acquired by AbGRI5 in a CR1-mediated event. Xie et al.22 detected the existence of a circular intermediate comprising blaPER-1-CR1-qacEΔ1/sul1, illustrating the importance of CR1 in the dissemination of blaPER-1. Like variants of AbGRI3, the right end of AbGRI5 (Figure 1b) contains a class 1 integron. However, the cassette array in AbGRI5, blaCARB-2-aadA2-cmlA1-aadA1, is different to the array seen in AbGRI3 variants, namely aacA4-catB8-aadA1.20 Upstream of the cassette array, the class 1 integron 5′-conserved segment (5′-CS) in AbGRI5 is interrupted by a fragment containing the ere(B) gene, in a configuration identical to that in Tn2610 (GenBank accession AB207867.1). Tn2610 contains part of Salmonella genomic island 1 (SGI1) (GenBank accession AF261825.2) and was generated by a series of recombination events.23 The integron and flanking sequences in AbGRI5 also appear to be the products of recombination events, as the cassette array likely comprises sequence derived from RJ458 on the left and from Tn2610 on the right (Figure 1c).
Chromosomal deletion mediated by IS26
In MDR-ZJ06, the gdhB gene is located in the position occupied by AbGRI5 in XH1056. One of the seven alleles used in the Oxford MLST scheme, gdhB, encodes a glucose dehydrogenase that catalyses the oxidation of glucose to gluconic acid.24 In XH1056, it appears that one of the copies of IS26 that bound AbGRI5 has deleted 16 232 bp of adjacent chromosomal sequence, removing several ORFs, including gdhB. Other ORFs lost in the deletion event encode a putative carbohydrate phosphorylation protein,25 a putative electrogenic bicarbonate transporter,26 a putative amino acid permease for amino acid uptake27 and a putative universal stress protein.28 Many instances of chromosomal loss or inversion mediated by IS26 have been reported in Acinetobacter,20,29 and such events are especially likely when the IS26 variant IS26-v3 is present,19 as it is in AbGRI3 and AbGRI5.
The deletion event in XH1056 was conspicuous as it removed an allele required for assignment of a complete Oxford scheme MLST profile. The loss of gdhB by XH1056 adds to previous reports of issues with this allele5,6 and suggests that the Oxford MLST scheme for A. baumannii might require revision. However, a simple solution to the issue presented by XH1056 might be to assign an absent gdhB allele as zero.
AbGRI5 was acquired as part of the GC2 chromosomal fragment
As the AbGRI3-like island AbGRI5 lies within the region of XH1056 chromosome that was acquired from a GC2 isolate, it is possible that the resistance island was acquired along with the chromosomal sequence in a single recombination event. However, AbGRI3 originally inserted in the putative GNAT family acetyltransferase gene atr,20 generating the 8 bp TSD AGGATGAG. Although some isolates carrying the resistance island have lost adjacent chromosomal regions in deletion events that have also removed the TSD, variants of AbGRI3 are always located in the same chromosomal region, which is not part of the segment of GC2 chromosome in XH1056. Thus, any putative donor GC2 isolate must have acquired AbGRI5 in a second event, such that it inserted in the chromosomal segment involved in the recombination event that yielded XH1056. The existence of such an isolate would indicate that GC2 A. baumannii has acquired AbGRI3-like resistance islands on two separate occasions.
It has been reported that XH859, a GC2 A. baumannii isolated in 2009 from a wound sample from a male patient in a hospital in Hubei, China30 contains an AbGRI3-like island, but has an intact atr gene, indicating that it might represent a second acquisition of an AbGRI3-like island by GC2.31 The XH859 chromosome (GenBank accession CP014539) was examined and the chromosomal region acquired by XH1056 was found to have been separated by an IS26-mediated inversion event. When this event was reversed in silico by inverting the 632 305 bp region (from position 1531841 to 2164145) flanked by inversely oriented copies of IS26-v3, an AbGRI3-like island was found between recA and gdhB, within the chromosomal fragment acquired by XH1056. Furthermore, the 24 936 bp island in XH859 contains blaPER-1 and is 99.98% identical to AbGRI5 of XH1056, but the class 1 integron appears to have been lost in an internal IS26-mediated deletion event of 9998 bp. A further deletion event mediated by the IS26 at the right end of AbGRI5 in XH859 has removed 1867 bp of adjacent chromosomal sequence relative to XH1056. As this deletion will also have removed the TSD associated with the insertion of AbGRI5, it is impossible to tell from the XH859 sequence precisely where AbGRI5 initially inserted. In order to determine this precise location, an isolate ancestral to XH859, with TSD intact, will need to be sequenced.
Although the precise insertion position of AbGRI5 in a sublineage of GC2 represented by XH859 could not be determined, it appears highly likely that an isolate related to XH859 is the source of the AbGRI5-containing chromosomal segment in XH1056.
Conclusions
Here, we have presented the complete genome of ST256 A. baumannii XH1056. Difficulties assigning XH1056 a profile with the Oxford MLST scheme prompted a detailed investigation of its chromosome. This revealed that the isolate is a hybrid derived from the integration via homologous recombination of an 18 kb stretch of A. baumannii GC2 chromosome, which contains the 34.9 kb resistance island AbGRI5. An IS26 in AbGRI5 is likely responsible for the deletion of a 16.2 kb stretch of adjacent chromosomal sequence, including gdhB. Finally, it was shown that AbGRI3 has been acquired by GC2 A. baumannii on two separate occasions and that the AbGRI5 and associated chromosomal sequence found in XH1056 were acquired from the rare GC2 lineage with AbGRI5 inserted near gdhB.
Funding
This work was supported by the grants from the National Natural Science Foundation of China (31970128, 81861138054, 31770142, 31670135), the Zhejiang Province Medical Platform (2020RC075) and the MRC-NSFC DETECTIVE project (MR/S013660/1). W.v.S. is supported by a Royal Society Wolfson Merit Award (WM160092).
Transparency declarations
None to declare.
References
EUCAST. Clinical Breakpoints; Breakpoint Table for Interpretation of MICs and Zone Diameters,
CLSI. Performance Standards for Antimicrobial Susceptibility Testing-Twenty—Ninth Edition: M100. 2019.
Author notes
Xiaoting Hua and Robert A. Moran contributed equally to this work, and should be considered as co-first authors.
