Introduction

Achromobacter spp. are gram negative rods capable of causing infections, including bacteremia, pneumonia and meningitis, mainly in immunocompromised patients. How-ever, infections in immunocompetent patients have also been reported^11,13. In addition, Achromobacter spp. are increasingly recognized as pathogens in patients with cys-tic fibrosis (CF). Different Achromobacter species, such as Achromobacter xylosoxidans, Achromobacter ruhlandii, Achromobacter dolens and Achromobacter insuavis, have been recovered from these patients, displaying variable fre-quencies among studies reported in different countries16. Members of this genus are naturally resistant to many antibiotics including ampicillin, cephalotin, aztreonam, cefotaxime, cefoxitin and aminoglycosides. The expression of multiple efflux pumps and the production of chromoso-mally encoded b-lactamases1 may be responsible for this resistance profile. Moreover, isolates displaying resistance to clinically relevant antibiotics such as colistin, carbapenem and fluoroquinolones have been described16. Little is known about the resistance markers involved in this acquired resis-tance profile. Thus, effective antimicrobial therapy can be challenging due to its inherent and acquired multidrug resis-tance patterns. Only a few plasmid-encoded b-lactamases have been identified in A. xylosoxidans, as is the case of VEB-115 and class B carbapenemases, such as blaIMP-1, blaIMP-10, blaVIM-11 and blaVIM-21. Moreover, plasmid descrip-tions are scarce in this genus, and no characterization of plasmids from A. ruhlandii have been reported, possibly due to misidentification of the species belonging to this genus.

Here we report the full characterization of a plasmid which was maintained in four clonally related A. ruhlandii isolates recovered from a single pediatric patient with CF, between 2013 and 2014.

Materials and methods

Nine Achromobacter spp. isolates were recovered from a pediatric patient with CF at a hospital in Buenos Aires between 2013 and 2015 (isolation dates are shown in Table 1). The isolates were cultured under aerobic conditions at 37 °C on TSA (Tryptic Soy Agar) and accu-rate identification was achieved by amplification and sequencing of the nrdA gene according to Spilker et al.18 XbaI-PFGE was performed according to Mireille-Cheron et al.5 to evaluate the clonal relationship between suc-cessive isolates. Minimum inhibitory concentration (MIC) values were determined for a representative set of antibi-otics: ampicillin, piperacillin, piperacillin/tazobactam, cef-tazidime, cefepime, imipenem, meropenem, ciprofloxacin, levofloxacin, kanamycin, gentamicin and colistin. The antimicrobial susceptibility test was assayed by the agar dilution method according to CLSI recommendations, using the breakpoints established for the category ''other non-Enterobacteriaceae”7. The Master pure DNA purification kit (Epicenter, Madison, WI, USA) was used for DNA extrac-tion in four selected isolates (Table 1). These 4 isolates were chosen because, in addition to being clonally related, they displayed differences in their antibiotic susceptibil-ity profiles regarding antimicrobial therapy in patients with CF.

Table 1: Antimicrobial resistance profiles of the tested isolates. 

Libraries were prepared for sequencing using the Illu-mina NexteraXT kit (Illumina Inc., San Diego, CA), and sequenced using paired-end reads of 150 base pairs in an Illumina NextSeq500 system at the Microbial Genome Sequencing Center. De novo assembly was performed with SPAdes v3.104, using a pre-assembly approach with Vel-vet v1.2.1021. All possible k-mer lengths were explored. The assembly was annotated using the RAST online server followed by manual curation. The presence of resistance determinants was assessed by Resfinder, MARA and CARD databases^10,20.

Plasmids from each isolate were assembled by contig overlapping. The comparison between plasmids was carried out using RAST, BLAST and the Artemis Comparison Tool. One of these plasmids was deposited in GenBank under AN: MK423762.1.

Plasmids from the A. ruhlandii isolates were extracted according to Kado and Liu12. Digestion with Kpnl was carried out to compare the restriction profiles. Moreover, PCR amplification of class 1 integron carrying the aac-(6')-Ib gene was performed6.

To assess their mobilization potential, plasmids were first introduced by electroporation into E. coli DH5a. Transfor-mants were selected in LBA medium containing 32 ^g/ml kanamycin. Afterwards, conjugation assays were carried out on LBA agar plates using the selected transformants as donor strainsand E. coli CAG12177as recipientcells. Different pro-portions of donor and recipient cells (10:1, 2:1, 1:1) were spread on agar plates and incubated overnight at 37 °C. Transconjugants were selected on agar plates containing 32 g/ml kanamycin and 32 g/ml tetracycline.

Results

Nine clonally related A. ruhlandii isolates were identi-fied from a single patient, based on the results of PCR amplification and sequencing of the nrdA gene, and XbaI-PFGE (data not shown). Different resistance profiles were observed among successive isolates. All isolates were resis-tant to ampicillin, cefepime, and aminoglycosides, which correlates with the intrinsic resistance profile observed in A. xylosoxidans, and to colistin. Almost all of them were intermediate or resistant to fluoroquinolones. Piperacillin, piperacillin/tazobactam and imipenem were the most active antibiotics. Nevertheless, one A. ruhlandii isolatewas resistant to piperacillin and piperacillin/tazobactam and fourout of nine A. ruhlandii isolates were categorized as interme-diate or resistant to imipenem (Table 1). According to the different susceptibility patterns observed, isolates 1, 2, 3 and 6 were selected for WGS.

All genomes showed a size of approximately 6 000 000 bp with 6000 coding sequences and a GC content of 67.7%. Different antibiotic resistance determinants were found in all 4 genomes corresponding to: 6 putative p-lactamases, 33 putative efflux pump coding genes, 1 aminoglycoside mod-ifying enzyme (aac-(6')-Ib), the dihydropteroate synthase and chloramphenicol O-acetyltransferase and mutations in gyrA, gyrB, parC, and parD. Among the detected p-lactamase ORFs no differences were observed among the compared isolates. These ORFs corresponded to: (i) blaOXA-258a previously reported as species-specific marker in A. ruhlandii15; (ii) blaaxc, recently described by Fleur-baaij et al.9 in A. xylosoxidans as a novel p-lactamase with carbapenemase activity; and (iii) one molecular class A and three class C non identified p-lactamases. Complete sequences for 2 RND efflux pumps (AxyABM and AxyXY-OprZ) were detected in the 4 genomes and no differences were observed among them. AxyABM displayed 91% iden-tity with that reported in A. xylosoxidans and 97% with that reported in A. ruhlandii17. The complete sequence for axyXY-oprZ coding genes displayed 96% identity with the previously described in A. xylosoxidans2,3. Twelve putative outer membrane coding genes were detected, being iden-tical in all isolates. No Pseudomonas aeruginosa OprD porin homologues were found.

With regard to plasmid characterization, the four iden-tified plasmids had a size of 34096bp with 50 coding sequences and an average G + C content of 63.3% (Supple-mentary Table 1).

These plasmids contained a mercury resistance operon region of 3351 bp containing merT, merR, merP, merA, merD, and merE; which were flanked by a Tn3 family transposase and the tra operon. In addition, it had a partition and replication region of 2217 bp comprising genes tnpR, repA, parA and an ATPase gene, which showed 99% coverage and 100% identity with an IncP6 plasmid found in P aeruginosa (CP033834.1). A transference region of 4923bp containing the trb (trbL, trbK, trbJ) and tra (traI, traJ, traK) loci was observed. These plasmids also contained a class 1 integron of 2835 bp carrying the aac-(6')-Ib gene, the relE/stbE toxin/antitoxin system, two copies of the dihydropteroate synthase gene, -one of them being truncated-, and one copy of the chloramphenicol-O-acetyltransferase (Fig. 1).

The four completely sequenced plasmids were almost identical. Only one of them showed one amino acid differ-ence in both dihydropteroate synthase coding genes. The presence of this plasmid was assumed in the other clonally related isolates, based on the identical KpnI restriction profiles and the presence of the aac-(6')-Ib gene located in the variable region of a class 1 integron displayed among them.

The overall backbone of the plasmids showed high homol-ogy (79% query cover and 99% identity) with P. aeruginosa plasmid pmatVim-7 (AM:778842.1), the tra and replication regions being almost identical as mentioned above (Supple-mentary Fig. 1).

The conjugation assays rendered negative results under the performed conditions.

Discussion

In this study we report nine clonally related A. ruhlandii isolates, recovered from the same patient. These iso-lates displayed different antibiotic resistance profiles, in concordance with previous reports in this species8. Vari-ations observed among these isolates suggest adaptive strategies that could lead to microevolution of resistance patterns in these patients. In silico analysis of A. ruhlandii genomes showed a great diversity of antimicrobial resis-tance determinants, which reveals the genomic plasticity of Achromobacter spp. and suggests that this genus could be a reservoir of antibiotic resistance markers. However, in the in silico comparative analysis no significant differences were observed among the 4 analyzed genomes, despite their different resistance profiles. Further studies on putative resistance markers identified in the whole genome analysis should be conducted to understand the resistance patterns observed in A. ruhlandii isolates.

Figure 1: Plasmid recovered from A.ruhlandii AN: MK423762. Overview and description. 

This is the first full description and characterization of a plasmid from A. ruhlandii. This plasmid was maintained in nine isolates recovered from the same patient. No significant mutations were found in silico among the four sequenced plasmids, indicating that this plasmid was preserved over time. Only an aminoglycoside resistance marker was found on this plasmid, indicating that mutations and/or variations in chromosomal gene expression should be involved in the resistant patterns observed in the isolates.

A comparative analysis of this plasmid with other 26 Achromobacter spp. available plasmids (A. xylosoxidans, Achromobacter insolitus, Achromobacter denitrificans, Achromobacter pestifer) indicated that the present plasmid from A. ruhlandii was not related with the others. The class 1 integron containing the aac-6'-Ib gene and the mercury resistance operon were detected in 6/26 studied plasmids. Only 2 plasmids corresponding to A. insolitus shared both the replicon and the addition system (relE/stbE), while 2 A. xylosoxidans plasmids harbored only the same replicon. The doc/phD toxin/antitoxin system was not observed in any of the studied plasmids.

Despite its high homology with pmatVim-7 from P. aeruginosa, blaVIM7 was not present in this plasmid. The metallo-carbapenemase coding gene was carried in a mobile element containing the tnpA15 gene, which may indicate that this element could have been either acquired within

P aeruginosa or lost once the plasmid was incorporated into A. ruhlandii14,19. This description provides new insights into horizontally acquired elements in A. ruhlandii, highlighting its capability of acquiring or transferring genes horizontally from/to other non-fermenters.

The conjugation experiments performed by Li et al. on P. aeruginosa were not successful under laboratory conditions, probably due to the lack of certain genes on the tra and trb regions, such as traF, traG, trbA and trbI14. However, a putative oriT was detected, which could make this plasmid mobilizable. These outcomes are also in accordance with our results, which showed the failure to render transconjugants under the assayed conditions, even if some genes of the tra region such as traR and traG that were missing on the plasmid were encoded within the genome. However, the traG gene was truncated and genes of the trb region such as trbP, trbN, and trbM were missing.

This study provides new insights into this emerging pathogen, which may contribute to develop the necessary knowledge to better understand the persistence in patients colonized/infected by this pathogen and likely contribute to a better management of its evolution.

Data availability

The plasmid sequence reported here has been deposited in GenBank under the accession number: MK423762.1.

Conflict of interest

The authors declare that they have no conflicts of interest.

C. Steffanowski, M. Papalia, A. Iriarte et al.

Funding

This research was supported by Universidad de Buenos Aires, Programación Científica UBACyT: 20020150100174BA (20162019) to Marcela Radice and by ANPCYT-PICT 1925-2015 to Gabriel Gutkind and by ANPCYT-PICT-2017-3996 to Mariana Papalia.