Distinct Mechanisms of Dissemination of NDM-1 Metallo-β-Lactamase in Acinetobacter Species in Argentina

A 4-year surveillance of carbapenem-resistant Acinetobacter spp. isolates in Argentina identified 40 strains carrying bla_NDM-1. Genome sequencing revealed that most were Acinetobacter baumannii, whereas seven represented other Acinetobacter spp. The A. baumannii genomes were closely related, suggesting recent spread. bla_NDM-1 was located in the chromosome of A. baumannii strains and on a plasmid in non-A. baumannii strains.

ABSTRACT

A 4-year surveillance of carbapenem-resistant Acinetobacter spp. isolates in Argentina identified 40 strains carrying bla_NDM-1. Genome sequencing revealed that most were Acinetobacter baumannii, whereas seven represented other Acinetobacter spp. The A. baumannii genomes were closely related, suggesting recent spread. bla_NDM-1 was located in the chromosome of A. baumannii strains and on a plasmid in non-A. baumannii strains. A resistance gene island carrying bla_PER-7 and other resistance determinants was found on a plasmid in some A. baumannii strains.

TEXT

The predominant species expressing the NDM-1 carbapenemase are Klebsiella pneumoniae and Escherichia coli. However, Acinetobacter spp. isolates are recognized as intermediate reservoirs for the bla_NDM-1 resistance determinant (1, 2). bla_NDM is a metallo-β-lactamase (MBL) generally found on a plasmid or other mobile element that carries resistance determinants for other antibiotic classes, rendering many NDM-positive isolates extensively drug resistant. Infections caused by carbapenem-resistant Acinetobacter baumannii isolates are associated with mortality rates as high as 60% (3, 4).

The Argentina National Reference Laboratory (NRL) identified an increase in the prevalence of NDM-containing Acinetobacter spp. isolates beginning in 2015. Of the 20,028 clinical isolates screened since 2010, 15,621 were carbapenem resistant and 144 had an MBL phenotype (5–7). PCR assays for common MBLs confirmed that 68 (47%), i.e., 40 bla_NDM and 28 bla_IMP, of 144 isolates were producers; whereas in the remaining strains, the MBL-like phenotype observed in the institution of origin was due to the presence of bla_OXA-23 or bla_OXA-58.

NDM-producing strains were recovered from 19 hospitals in nine cities and seven provinces in Argentina (see Fig. S1 in the supplemental material). A. baumannii isolates had more extensive antimicrobial resistance profiles than the non-baumannii isolates (see Table S1 in the supplemental material). Genome sequences were obtained on an Illumina NextSeq 500 and assembled using Velvet (8). A BLASTN search at NCBI classified seven genomes as representing five different non-baumannii Acinetobacter species. (see Table S1). MLST analysis showed that all 33 A. baumannii isolates belonged to sequence type ST25 (9). The ST25 genomes were most closely related to isolates found throughout the world, including HEU3 (Honduras), HWBA8 (South Korea), NM3 (United Arab Emirates), and two genomes with no geographic origin provided, i.e., AR_0088 and AB5256. The AR_0088 genome has been completely sequenced (GenBank accession no. CP027530.1) and was used as the reference genome for single nucleotide polymorphisms (SNPs) and insertion element (IS) annotation.

The A. baumannii genomes differed by only 14 to 36 sequence variants, suggesting recent divergence. Patterns of shared SNPs (10), IS locations (11), and epidemiological data were highly concordant (Fig. 1). Genomes were predominantly clustered by isolation location, with a few exceptions. For example, AMA19 from Hospital General de Agudos Vélez Sarsfield (VELE) was essentially identical to AMA9 from Hospital Dr. Cosme Argerich (COS) and on the same branch as other COS isolates. These hospitals belong to the public care sector in the Buenos Aires capital district, where patient exchange is frequent, suggesting a possible transmission event.

FIG 1.

Phylogenetic tree of A. baumannii genomes. A neighbor-joining tree was constructed using shared SNP and IS insertion sites. Inner circle shows year of isolation. Outer circle shows hospital in which the isolate was recovered. See Table S1 for further information on the location of each hospital. Isolates that are positive for components of RI-PER-7 are denoted by colored circles outside of the tree. The scale bar represents the combined number of SNPs and IS insertion events. The AR_0088 genome was used as the out group, but the branch length is not shown to highlight the relationships of the AMA strains. The figure was created using iTOL (24).

The AR_0088 reference genome contained two plasmids, i.e., pAR_0088_1 (GenBank accession no. CP027531.1) and pAR_0088_2 (GenBank accession no. CP027532.1). pAR_0088_2 carries the bla_NDM-1 gene in Tn125, and this plasmid is the likely location of the bla_NDM-1 gene in the non-baumannii Acinetobacter genomes (1, 12, 13). pAR_0088_2 sequences were not present in the A. baumannii ST25 genomes. To ascertain the location of Tn125 in the A. baumannii genomes, we identified the locations of ISAba125 insertions, which flank the transposon, in the draft genome assembly of a representative strain (AMA16). Six ISAba125 insertion sites were inferred, all in the chromosome, based on alignment of flanking sequences to the AR_0088 reference sequence. PCR and Sanger sequencing were used to demonstrate that Tn125 was inserted at base 3,921,386 of the AR_0088 genome in AMA16 and all other A. baumannii isolates, interrupting a gene encoding a hypothetical protein, AM467_RS18915 (see Table S2 in the supplemental material).

A large resistance gene island (RI) was identified in 14 of the ST25 strains (RI-PER-7). This ∼23.8-kb sequence is bound by a pair of IS26 elements in direct-repeat orientation (Fig. 2). The island carries genes encoding resistance to aminoglycosides (armA), rifampin (arr), cephalosporins (bla_PER-7), and fosfomycin (GST) (Fig. 2). This RI also carries two copies of ISCR1 (IS91-like) and three other IS elements that are found predominantly in Enterobacteriaceae isolates but are rare in Acinetobacter isolates, i.e., IS10A, ISEc28, and ISEc29. The 7.5 kb at the 3′ end of the island is found in several non-Acinetobacter genomes. The complete structure was identified in seven accessions in GenBank, mostly plasmid sequences. Five of the ST25 genomes harbored a shorter version of the RI, lacking the bla_PER-7 gene. Genetic structures similar to RI-PER-7 were identified in other species, including K. pneumoniae, Proteus mirabilis, and E. coli. In these structures, bla_PER-7 was not present, but other β-lactamases were found (Fig. 2).

FIG 2.

Comparison of genetic structure of RI-PER-7 genomic island. Gray bars, regions shared between isolates; red arrows, IS elements; green arrows, antibiotic resistances genes. The figure was created using EasyFig, version 2.2.2.

RI-PER-7 is not present in AR_0088, but it was found on one of the plasmids in HWBA8 (14). pHWBA8_1 (GenBank accession no. CP020596.1) is 195,838 bases, and a large portion of this sequence is present in the ST25 A. baumannii genomes, including the complete RI-PER-7. Interestingly, pHWBA8_1 is also similar to pAR_0088_1, but the latter plasmid lacks the RI and another ∼17.5-kb segment and has extensive rearrangements covering another ∼20 kb of the sequence. The location of RI-PER-7 in the plasmid was confirmed by PCR amplification and Sanger sequencing of the junction regions (see Table S2). This large plasmid carries five additional AR genes located outside RI-PER-7.

The comM gene, a common location for insertion of RIs in A. baumannii isolates, is intact in ST25 strains. IS elements were not found upstream of the chromosomal bla_ADC or bla_OXA genes in this study. The ST25 strains carry S84L and S81L substitutions in the parC and gyrA genes, consistent with their nonsusceptibility to ciprofloxacin.

The NRL also confirmed the presence of bla_NDM-producing Enterobacteriaceae in 12 of 19 hospitals (data not shown). In 8 of the 12 hospitals, this emergence occurred 1 to 4 months after the first detection of NDM-producing Acinetobacter isolates. Additional work is needed to determine whether Acinetobacter spp. could have played a role in the interspecies dissemination of NDM in these institutions.

Although reports of bla_NDM in Latin America are limited, sentinel investigations have described the presence of Tn125 in Acinetobacter spp. (6, 15–17). An ST25 A. baumannii strain from a patient with an abdominal infection in Honduras was determined to have Tn125 on a plasmid (18), but this plasmid was not present in the ST25 strains analyzed here. Tn125 has been reported in the chromosome of Acinetobacter spp. as well (19, 20).

ST25 strains in Argentina typically carry bla_OXA-23 β-lactamase (17). According to available data, the cooccurrence of bla_NDM-1 and bla_OXA-23/58 seems to be an uncommon event (12, 21–23), a scenario that may change due to the increasing emergence of NDM-1 in A. baumannii isolates.

In summary, the genetic context of bla_NDM-1 differs between A. baumannii and non-baumannii isolates in Argentina, with non-baumannii strains mostly retaining susceptibility to some antibiotics. We also describe an RI in a subset of A. baumannii genomes that likely contributes substantially to the MDR phenotype of these strains. The escalating number of reports of NDM-1 among A. baumannii isolates suggests a switch regarding the genetic basis of carbapenem resistance in this species, and intensive tracking of patient contacts is warranted for ST25.

Data availability.

This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under BioProject accession no. PRJNA562922. Contig sequences for each genome are available under GenBank accession nos. VYSH00000000 to VYTU00000000.