Plasmid-borne mcr-1 and replicative transposition of episomal and chromosomal blaNDM-1, blaOXA-69, and blaOXA-23 carbapenemases in a clinical Acinetobacter baumannii isolate

Abstract

A multidrug-resistant clinical Acinetobacter baumannii isolate with resistance to most antibiotics was isolated from a patient at an intensive care unit. The genetic environment, transcriptome, mobile, and resistome were characterized. The MicroScan system, disc diffusion, and broth microdilution were used to determine the resistance profile of the isolate. A multiplex PCR assay was also used to screen for carbapenemases and mcr-1 to -5 resistance genes. Efflux-pump inhibitors were used to evaluate efflux activity. The resistome, mobilome, epigenome, and transcriptome were characterized. There was phenotypic resistance to 22 of the 25 antibiotics tested, intermediate resistance to levofloxacin and nalidixic acid, and susceptibility to tigecycline, which corresponded to the 27 resistance genes found in the genome, most of which occurred in multiple copies through replicative transposition. A plasmid-borne (pR-B2.MM_C3) mcr-1 and chromosomal bla_PER-7, bla_OXA-69, bla_OXA-23 (three copies), bla_ADC-25, bla_TEM-1B, and bla_NDM-1 were found within composite transposons, ISs, and/or class 1 and 2 integrons on genomic islands. Types I and II methylases and restriction endonucleases were in close synteny to these resistance genes within the genomic islands; chromosomal genomic islands aligned with known plasmids. There was a closer evolutionary relationship between the strain and global strains but not local or regional strains; the resistomes also differed. Significantly expressed/repressed genes (6.2%) included resistance genes, hypothetical proteins, mobile elements, methyltransferases, transcription factors, and membrane and efflux proteins. The genomic evolution observed in this strain explains its adaptability and pandrug resistance and shows its genomic plasticity on exposure to antibiotics.

Importance: A pandrug-resistant pathogen that was susceptible to only a single antibiotic, tigecycline, was isolated from a middle-aged patient in an ICU. This pathogen contained two plasmids and had a chromosome that contained portions that were integrated externally from plasmids. These genomic islands were rich with resistance genes, mobile genetic elements, and restriction-modification systems that protected the pathogen and facilitated gene regulation. The strain contained 35 resistance genes and 12 virulence genes. The strain was of closer evolutionary distance to several international strains suggesting that it was imported into South Africa. However, its resistome was unique, suggesting an independent evolution on exposure to antibiotic therapy mediated by epigenomic factors and MGE transposition events. The varied mechanisms available to this strain to overcome antibiotic resistance and spread to other areas and/or transfer its resistance determinants are worrying. This is ultimately a risk to public health, evincing the need for antibiotic stewardship.

Keywords: RNAseq; carbapenem; carbapenemase; colistin resistance; last-resort antibiotics; multi-drug resistance; non-fermenters.

Fig 1

Genetic environment of blaOXA-23 carbapenemase on chromosomal contig 1 (1.19–1.22 Mb, 1.23–1.28 Mb). Mobile genetic elements (shown as red arrows) and methylases/restriction modification endonuclease (purple arrows) bracketing the bla_OXA-23 gene show that the gene is within a composite transposon. The same chromosomal contig has two bla_OXA-23 genes shown in panels A and B, with the genetic environment in panel A being different from that in panel B. In both cases, bla_OXA-23 was bracketed by ISAba1 and transposases.

Fig 2

Genetic environment of sul2, aph(3”)lb, aph(6)-ld, and blaOXA-23 and blaNDM-1 carbapenemase on chromosomal contig 1 (~1.32–1.34 Mb, ~320–360 kb).bla_OXA-23 (shown as blue arrow) in panel A, is bracketed by a composite transposon consisting of ISAba1 and transposases. sul2, aph(3″)lb, aph(6)-ld, and bla_NDM-1, in panel B (shown as red arrows), were sandwiched between IS1006, recombinase, ISAba1, IS30, and ISVsa3 insertion sequences and transposases.

Fig 3

Genetic environment of aph(3′)-la, aac(3)-la, aadA1, sat2, dfrA, and Sat2 resistance genes on chromosomal contig 1 (~450–500 kb, 2.04–2.06 Mb, ~2.375–2.39 Mb). IS26-IS6 and ISPpu12 transposase bracketed aph(3’)-la, aac(3)-la, and aadA while a recombinase and class 1 integron integrase (IntI1) sandwiched sul1, qacE, GNAT family N-acetyltransferase, and aadA1 genes. Resistance genes are shown as blue arrows and mobile genetic elements are shown as red arrows. A DNA adenine methylase (purple arrow) was also found in close proximity to the resistance genes within the same genomic island on chromosome (A) N-acetyl transferase and bla_OXA-51 carbapenemase were also found in close proximity to trmA methylase (purple arrow) within the same region without any mobile genetic element (B) dfrA, sat2, and aadA1 are also bracketed by a class 2 integron (IntI2), IS256 and TnsD/E (Tn-7-like) transposases (C).

Fig 4

Genetic environment of aadA1, sat2, dfrA, and blaADC resistance genes on chromosomal contig 2 (0–15 kb, 710–720 kb, and 1.8Mb–1.82 Mb). IS256, TnsE, and tnsD mobile genetic elements (shown in red arrows), recombinase (orange arrow), and an endonuclease (purple arrow) bracketed dfrA, sat2, aadA (A) while bla_ADC was in close synteny with ISAba1 (B) The region shown in panel C is a “cut and paste” transposition of the region shown in panel A but in the reverse orientation, suggesting that a replicative transposition event occurred within the genome.

Fig 5

Genetic environment of mcr-1, tet(M), QnrS1, dfrA15, blaTEM-135, tet(A), cmlA1, aadA, qaC, and sul3 resistance genes on plasmid pR-B2.MM_C3 (contig 3). The resistance genes (blue arrows) were clustered together on a genomic island on pR-B2.MM_C3 at 0–42kb and 155 kb-0kb and sandwiched between composite transposons (red arrows) and integrons (orange arrows). The mobile genetic environment is comprised of composite transposons such as IS903B, IS26, IS1380, IS1A, and Tn3, alongside recombinases and class 1 IntI1 integron. Site-specific DNA methylases were also found within this genomic island.

Fig 6

Phylogenetic analysis of antibiotic-resistant Acinetobacter baumannii strains from Africa. The R-B2.MM strain was not closely related to any resistant strain in Africa. The most closely related strains (A. baumannii 13259 and 13367) were not supported by the bootstrap values to be significant. The R-B2.MM strain is shown as red while all other strains are shown as black. Bootstrap values of ≥50 is significant. The resistomes of the three strains are also shown in a table below the tree, with A. baumannii 13367 having more similarity with this study’s strain. MLST (1) is the Pasteur Institute typing scheme while MLST (2) is the PubMLST typing scheme.

Fig 7

Phylogenetic analysis of antibiotic-resistant global Acinetobacter baumannii strains. A. baumannii Ab905 (from Tel-Aviv, Israel, in 2019), Ab241 (from Tel-Aviv, Israel in 2019), Ab238 (from Tel-Aviv, Israel, in 2019), AbCTX19 (from Le Kremlin Bicetre, France, in 2019), and A3232 (Greece, 2022) strains formed the same clade with the R-52.MM strain. All these strains were isolated from humans and mostly from blood except strain AbCTX19 (rectal swab). Ab241 and Ab238 were of the same MLST (106 or 3) while AbCTX19 and A3232 were of MLST 231 (and 1 or 160, respectively). The significance of the clade was confirmed by the bootstrap. A3232 was most closely related to R-B2.MM evolutionarily and is thus shown as red on the tree. Bootstrap values of ≥50 is significant. MLST (1) is the Pasteur Institute typing scheme while MLST (2) is the PubMLST typing scheme.

Fig 8

A volcano plot showing differentially expressed genes (DEGs) in Acinetobacter baumannii R-B2.MM was exposed to colistin and carbapenems. The significant DEGs are shown in red while the non-significant DEGs are shown in gray. The significant DEGs include genes within the category of hypothetical protein, LysR transcriptional regulator, phage replication protein, aliphatic sulfonate monooxygenase, aldehyde dehydrogenase. Putative lipoprotein, urea carboxylase-related aminomethyltransferase, ribulose-5-phosphate 4-epimerase, and VgrG protein. The annotation on the plot shows highly expressed genes such as ABC efflux (E) OMP W (outer membrane protein W), int1 (integrase/class 1 integron), resistance genes (sul-2, bla_NDM, bla_PER, GNAT acetyl transferase), ISs (insertion sequences, transposons), and type VI secretion systems (T6SS). Highly repressed genes included Type I RMS I, outer membrane porins (OprD) and proteins (OMP), prophage elements, acrR transcriptional regulators, and ABC and MFS efflux pumps.