Comparative Genomics of Two ST 195 Carbapenem-Resistant Acinetobacter baumannii with Different Susceptibility to Polymyxin Revealed Underlying Resistance Mechanism

Abstract

Acinetobacter baumannii is a Gram-negative nosocomial pathogen of importance due to its uncanny ability to acquire resistance to most antimicrobials. These include carbapenems, which are the drugs of choice for treating A. baumannii infections, and polymyxins, the drugs of last resort. Whole genome sequencing was performed on two clinical carbapenem-resistant A. baumannii AC29 and AC30 strains which had an indistinguishable ApaI pulsotype but different susceptibilities to polymyxin. Both genomes consisted of an approximately 3.8 Mbp circular chromosome each and several plasmids. AC29 (susceptible to polymyxin) and AC30 (resistant to polymyxin) belonged to the ST195 lineage and are phylogenetically clustered under the International Clone II (IC-II) group. An AbaR4-type resistance island (RI) interrupted the comM gene in the chromosomes of both strains and contained the bla OXA-23 carbapenemase gene and determinants for tetracycline and streptomycin resistance. AC29 harbored another copy of bla OXA-23 in a large (~74 kb) conjugative plasmid, pAC29b, but this gene was absent in a similar plasmid (pAC30c) found in AC30. A 7 kb Tn1548::armA RI which encodes determinants for aminoglycoside and macrolide resistance, is chromosomally-located in AC29 but found in a 16 kb plasmid in AC30, pAC30b. Analysis of known determinants for polymyxin resistance in AC30 showed mutations in the pmrA gene encoding the response regulator of the two-component pmrAB signal transduction system as well as in the lpxD, lpxC, and lpsB genes that encode enzymes involved in the biosynthesis of lipopolysaccharide (LPS). Experimental evidence indicated that impairment of LPS along with overexpression of pmrAB may have contributed to the development of polymyxin resistance in AC30. Cloning of a novel variant of the bla AmpC gene from AC29 and AC30, and its subsequent expression in E. coli also indicated its likely function as an extended-spectrum cephalosporinase.

Keywords: Acinetobacter baumannii; carbapenem-resistant; polymyxin; resistance island; resistance mechanisms; whole genome sequencing.

Figure 1

Phylogenetic analysis of A. baumannii AC12, AC29, AC30, and completed A. baumannii genomes obtained from NCBI. The phylogenetic tree was constructed by composition vector tree version 2 (CVTree 2) using concatenated nucleotide sequences of the seven reference genes based on the Bartual/Oxford MLST scheme (cpn60, gdhB, gltA, gpi, gyrB, recA, and rpoD). Strains belonging to the international clonal groups IC-I, IC-II, and IC-III are indicated. The accession nos. of the strains used in the analysis are as follows: AB307-0294 (CP001172.1), AYE (CU459141.1), AB0057 (CP001182.1), ACICU (CP000863.1), 1656-2 (CP001921.1), TCDC-AB0715 (CP002522.2), BJAB0868 (CP003849.1), BJAB07104 (CP003846.1), M1 (LAIL01000001.1), TYTH-1 (CP003856.1), MDR-TJ (CP003500.1), MDR-ZJ (CP001937.1), OIFC137 (NZ_AFDK01000002.1), SDF (NC_010400.1).

Figure 2

Structures of resistance islands present in A. baumannii AC12, AC30, and AC29 as compared to similar islands in A. baumannii MDR-ZJ and MDR-TJ. (A) Structure of resistance island ACRI12-1 present in A. baumannii AC12. ΔTn6022, Tn6022, and Tn2006 are indicated in the red dotted-line box. Directions of genes and ORFs are indicated by arrows above the central thick line and the names are as given above. Green boxes represent truncated ΔcomM, turquoise blue boxes represent insertion element, ISAba1, and orange boxes represent genes conferring antibiotic resistance. Numbered boxes in gray represent genes encoding for hypothetical proteins. Comparison between structures of (A) AC12-RI1, (B) AC30-RI1, (C) AC29-RI1, (D) AbaR22, and (E) RI_MDR−TJ, revealed similarities in the islands, as indicated by the same colors and red dotted-line box.

Figure 3

Linear map showing the genetic organization of plasmids pAC29a, pAC30a, and pAC12 in comparison with other similar A. baumanii plasmids. The XerC/XerD-like recombination sites are indicated in black rectangular boxes with the nucleotide sequences as indicated above the boxes. The two plasmid replication genes, repB and repA, are indicated in blue whereas the putative abkA/abkB toxin-antitoxin genes are indicated in green arrows. Hypothetical ORFs are shaded gray. The TonB-dependent receptor gene is indicated in yellow whereas the putative septicolysin gene is in orange.

Figure 4

Linear map comparison of the Tn1548::armA region in plasmid pAC30b from A. baumannii AC30 and pMDR-ZJ06 from A. baumannii MDR-ZJ06 (NC_017172). The Tn1548::armA region and insertion elements (IS) were underlined and indicated in the diagram.

Figure 5

Circular map of the ~70 kb plasmids pAC30c (A) and pAC29b (B). Outer circle of the map represents ORFs found in the plasmid, gray ORFs are genes encoding for hypothetical proteins. The two inner circles represent GC plot and GC skew, whereby the green circle stands for above average and black circle stands for below average G+C content. Red colored arrow represents plasmid replication gene, rep; orange colored arrow represents resistant determinants; pink colored arrow represents tra genes; black colored arrow represents genes encoding proteins with known functional homologs; purple colored arrow represents putative toxin gene homologs of toxin-antitoxin systems; and gray colored arrow represents genes encoding hypothetical proteins.

Figure 6

Relative expression levels of pmrAB of the polymyxin-resistant A. baumannii AC12 and AC30 as compared to the polymyxin-susceptible A. baumannii AC29 as determined by Quantitative Real-Time reverse transcriptase-PCR (qRT-PCR). The data represents the mean fold change ± standard deviation (SD; indicated as error bars in the graph) taken from three replicates performed for each qRT-PCR reaction. Asterisk (^*) indicate statistical significance, as determined by using two-tailed, unpaired Student's t-test with p < 0.05.

Figure 7

SDS-PAGE analysis of the extracted lipopolysaccharide (LPS) layer of the A. baumannii ATCC19606 (control; polymyxin susceptible strain), AC12 (polymyxin resistant strain), AC29 (polymyxin susceptible strain), and AC30 (polymyxin resistant strain). M stands for the protein marker Precision Plus Protein Dual Xtra Standards (BioRad) with the sizes as indicated in kDa.