Acinetobacter baumannii is a successful pathogen characterized by its ability to acquire multidrug resistance (MDR) and to cause nosocomial outbreaks. Resistance to carbapenems in A. baumannii has increased in recent years and has been related to the production of carbapenemases, alterations in porins, overexpression of efflux pumps and altered penicillin-binding proteins.1 The main mechanism of resistance to carbapenems in A. baumannii is the acquisition of genes coding for carbapenem-hydrolyzing class D beta-lactamase (CHDL), also known as oxacillinases (blaOXA-23, blaOXA-24/40, blaOXA-58, blaOXA-143 and blaOXA-235).2 The role of the naturally occurring chromosomic blaOXA-51-like oxacillinases (i.e. blaOXA-51, blaOXA-66, blaOXA-69, blaOXA-71, blaOXA-94) in the resistance to carbapenems is unclear. Class B beta-lactamases (metallo-beta-lactamases; MBL) (i.e. blaVIM-like) are generally much less frequent than oxacillinases in carbapenem-resistant A. baumannii. Nevertheless, MBLs tend to hydrolyze carbapenems more efficiently than oxacillinases and have a broader spectrum of hydrolytic activity (including expanded-spectrum cephalosporins and aztreonam).2,3 Some of these MBLs, like the New Delhi metallo-β-lactamase-1 (NDM-1), are rapidly spreading worldwide in gramnegative bacteria, which represent a serious problem from a clinical and an epidemiological point of view.4,5 In A. baumannii, the gene encoding NDM-1 has been detected on the chromosome and, less frequently, in plasmids from isolates from different countries around the world.6 Since this is the first time that NDM-1-producing A. baumannii has been detected in Spain, we decided to (i) characterize this isolate; (ii) determine the genetic localization (chromosomal or plasmidic) of blaNDM-1 and (iii) analyze its genetic environment.

The isolate (Ab-NDM-1) was identified by MALDI-TOF MS and the SpeciesFinder 1.2 tool as A. baumannii, and was positive for blaOXA-51 by PCR. MICs obtained by microdilution using Type 44 panels are shown in Table 2. Etest MICs were >32mg/L for imipenem and meropenem, and 1mg/L for tigecycline. The isolate was susceptible only to amikacin according to EUCAST and CLSI breakpoints of 2018.12,13 The MIC of colistin was >64mg/L using the UMIC kit.

The isolate was assigned to ST85 by MLST and the MLSTFinder 1.8 tool, and was clonally unrelated (more than 5 band differences) to previous pulsotypes of carbapenem-resistant A. baumannii in the Andalusian database of the PIRASOA program reference laboratory. Synergy was observed between the meropenem disks and the dipicolinic acid disks. blaOXA-51-type and blaNDM-type genes were detected by endpoint PCR, and were identified as blaOXA-94 and blaNDM-1, respectively, by Sanger sequencing.

After trimming, 578,845 reads were obtained by WGS. The average length of these reads was 260.3bp and GC content was 39.5%. After assembly, the draft genome consisted of 316 contigs, with mean length of 22,375bp and mean sequencing coverage of 53, yielding a total read length of 3,950,783bp. After genome annotation using Rapid Annotations using Subsystems Technology (RAST) server (http://rast.theseed.org/FIG/rast.cgi), a total of 3703 coding sequences and 78 RNAs were obtained.

The acquired antimicrobial resistance genes identified in the assembled contigs with the ResFinder 1.2 tool were associated with resistance to aminoglycosides (aadB, which confers resistance to gentamicin and tobramycin), beta-lactams (chromosomal cephalosporinase blaADC-25, class B carbapenemase blaNDM-1 and the OXA-51-type CHDL, blaOXA-94), macrolides, lincosamides and streptogramins [msr(E), mph(E)], phenicols (floR) and sulfonamide (sul2). The resistome for the most relevant group of antimicrobials is shown in Table 2. WGS revealed that blaNDM-1 was located on a defective transposon (ΔTn125) flanked by the insertion sequences ISAba125 and ISAba14 inserted in the upstream and downstream regions, respectively (Fig. 1).

Fig. 1.

Schematic representation of the genetic environment of blaNDM-1: ble gene, encoding the bleomycin resistance protein; iso gene, encoding a putative phosphoribosylanthranilate isomerase; tat gene, encoding oxidoreductase DsbD superfamily protein; dct, gene, encoding divalent cation tolerance protein.

(0.04MB).

With respect to chromosomal mechanisms related to carbapenem resistance, mutations in the porin genes, carO and oprD-like, were not detected, whereas several point mutations (V90I, H127K, S168G, T173S, D183N, S236T, S243E and V270L) were observed in the omp33-36 gene. No mutations were detected in genes coding for the two-component regulatory system adeR-adeS, which regulates the expression of genes encoding the efflux pump adeABC.

With respect to colistin resistance, acquired resistance genes (mcr-1) or mutations in the chromosomal genes pmrA, lpxA, lpxC and lpxD were not detected. Table 2 shows the aminoacid changes detected in pmrB and pmrC.

The isolate carried three different plasmids, which were ∼150kb, ∼37kb and ∼6kb in size, respectively. No transconjugants or transformants producing NDM were obtained. The blaNDM-1 probe hybridized to chromosomal DNA, but not to any of the three plasmids observed.

This draft genome project has been deposited registered at GenBank (accession number QBY00000000.1) with the BioProject ID PRJNA449628 (http://www.ncbi.nlm.nih.gov/bioproject/449628).

In countries all over the world, the acquired carbapenemase NDM-1 is spreading efficiently in several gramnegative microorganisms in nosocomial and community-acquired infections, which poses a major challenge for the treatment and control of healthcare-associated infections.4,5,14,15 In A. baumannii, this MBL has been detected in carbapenem-resistant isolates in different countries of Europe (Switzerland, Slovenia, Germany, France, Belgium, Czech Republic, Turkey), Latin America (Colombia), Africa (Algeria, Ethiopia, Syria,) and Asia (India).15–21 In Spain, NDM-1-producing A. baumannii has not been reported before.

Isolate Ab-NDM-1 showed cross-resistance with other antimicrobial families (quinolones and some aminoglycosides) consistent with other NDM-1-producing isolates in other countries. This reduces the therapeutic options for infection to certain toxic antimicrobials such as colistin.14 Although this isolate lacked some of the most frequently described CHDL genes in A. baumannii (OXA-23-like, OXA-24/40-like, OXA-58-like), carbapenem resistance is fully explained by the acquisition of blaNDM-1. Nevertheless, additional carbapenem resistance mechanisms, probably related to the Omp 33–36 porin, may be implicated, as described in A. baumannii isolates in other studies.22–24 Although some of the amino acid changes observed in Omp 33–36 of Ab-NDM-1 could be associated with decreased susceptibility to carbapenems, additional studies with isogenic mutants are required to understand their role in carbapenem resistance.

The absence of mutations in the regulatory genes adeS and adeR suggests that the efflux pump adeABC, implicated in A. baumannii resistance to some antimicrobials and biocides, is not overexpressed in Ab-NDM-1, which is in agreement with the absence of resistance to tigecycline.25,26

Isolate Ab-NDM-1 also showed resistance to colistin, which was associated with some aminoacid changes in pmrC and particularly in pmrB (i.e. A227V). The implication of other less frequent or undescribed mechanisms of colistin resistance related to the synthesis of lipid A cannot be ruled out.27

In Enterobacteriaceae, blaNDM-1 is frequently plasmid encoded, whereas it is more frequently found on the chromosome in A. baumannii, although it has also been detected on plasmids.6,15,16 Similar genetic environments have been detected harboring blaNDM-1 in Enterobacteriaceae and A. baumannii, indicating horizontal transmission (plasmid transfer) of this carbapenemase, probably from Enterobacteriaceae to A. baumannii.28

The carbapenemase blaNDM-1 was detected on the chromosome of Ab-NDM-1, but not in any of the three plasmids observed. This finding suggests that the clone must have been introduced into the Hospital Puerta del Mar by clonal dissemination, rather than by plasmid transfer from a NDM-1-producing gramnegative isolate not previously detected in this center. Another possible hypothesis that was not investigated in this study is phage-mediated transfer of blaNDM-1, as has been described among isolates of A. baumannii.6

Tn125-like transposons are the most frequent genetic platforms for mobilization of blaNDM-1.15 In Ab-NDM-1 isolates, the blaNDM-1 carbapenemase is associated with a truncated transposon (ΔTn125) containing a genetic environment similar or identical to those previously described in other carbapenem-resistant NDM-1- and OXA-94-producing A. baumannii isolates, particularly those belonging to the ST85 clone previously detected in some countries (France, Algeria, Turkey, Syria, Tunisia) (19–22,30).18–21,29 In Spain, the A. baumannii ST85 clone has only been detected in OXA-23-producing carbapenem-resistant isolates causing nosocomial outbreaks.30

Tn125-like transposons have also been detected in A. baumannii clones other than ST85, such as clone ST25 (in Slovenia, Germany, Lebanon), which is not related to CC92/CC2, but included in a different CC together with the ST6 clone belonging to international clonal lineage I (in Switzerland).18–21

The patient with Ab-NDM-1 made no reference to any history of travel to other countries where NDM-1-producing bacteria are prevalent, so that the source of this Ab-NDM-1 isolate in the Hospital Puerta del Mar remains unclear. At the same time, contact between this patient and other patients or healthcare personnel colonized with NDM-1-producing gramnegative microorganisms cannot be ruled either.

This study has some limitations. One of them is the lack of epidemiological and clinical data about other patients admitted to the same center, which makes difficult to investigate potential reservoirs and transmission routes, or the country of origin of the Ab-NDM-1 isolate.

In conclusion, to the best of our knowledge, this is the first time that blaNDM-1 has been detected in blaOXA-94-producing carbapenem-resistant A. baumannii belonging to the ST85 clone in Spain.

This work was partially funded by a grant (PI11/02046) from the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad.

None to declare.