Characterization of the Diversity in Host Range of an Extensively Drug-Resistant (XDR) Type IV Secretion System-Encoding Plasmid in Acinetobacter

Abstract

The World Health Organization (WHO) cites antimicrobial resistance as among the greatest threats to human health. The multidrug-resistant pathogen Acinetobacter baumannii, recognized as a priority pathogen for healthcare and research, is responsible for a diverse array of infections including respiratory tract, soft tissue and wound, and bloodstream infections. Despite this importance, the mechanisms of its pathogenesis remain poorly understood. Conjugation represents a central mechanism for bacterial adaptation and evolution and is responsible for the spread of genes that promote pathogen survival, antibiotic resistance, virulence, and biofilm formation. Our laboratory recently characterized a large group of almost 120 Type IV Secretion System (T4SS)-encoding plasmids in Acinetobacter, distributed globally across 20 countries spanning four continents, and demonstrated that an XDR A. baumannii plasmid from this family was transmissible to another A. baumannii strain. This research investigated the potential diversity of host strains for this representative member plasmid. Using the GC1 lineage strain A. baumannii AB5075-UW harbouring the XDR plasmid p1AB5075 and a series of previously characterized clinical and environmental Acinetobacter strains, conjugative analyses demonstrated transfer of the XDR plasmid to both A. baumannii strains of more genetically divergent sequence types and to non-baumannii Acinetobacter species both inside and outside the Acinetobacter calcoaceticus-baumannii (ACB) complex. Successful recipients included diverse strains of both clinical and environmental origin within the Acinetobacter genus. Collectively, this research could provide insights into an important genetic element for future surveillance.

Keywords: Acinetobacter baumannii; antibiotic resistance; bacterial pathogen; carbapenem resistance; conjugation; horizontal gene transfer; secretion system.

Figure 1

Conjugative transfer of the XDR plasmid p1AB5075 to environmental A. baumannii strains. (a,b) Diagnostic PCR analysis of the p1AB5075 T4SS-encoding gene cluster (a) and antibiotic resistance gene cluster (b).

Figure 2

Conjugative transfer of the XDR plasmid p1AB5075 to more genetically divergent Acinetobacter species. (a,b) Diagnostic PCR analysis of the p1AB5075 T4SS-encoding gene cluster (a) and antibiotic resistance gene cluster (b).

Figure 3

Assessment of p1AB5075 antibiotic resistance gene region in A. baumannii AB046 transconjugants. (a) The antibiotic resistance gene region of p1AB5075. Antibiotic resistance genes are shown in colour and genes not associated with antibiotic resistance are shown in grey. The resistance genes of interest, ant(2″)-Ia, aac(6′)-Ib10, and bla_GES-11, are shown in cyan, light green, and blue, respectively, while all other resistance genes are denoted in light purple. (b,c) Diagnostic PCR analysis of p1AB5075 antibiotic resistance gene cluster. (b) Region containing ant(2″)-Ia. (c) Region containing bla_GES-11 and aac(6′)-Ib10.

Figure 4

Cefotaxime inactivation by Acinetobacter transconjugants harbouring XDR plasmid p1AB5075. (a) Schematic diagram of cefotaxime inactivation. Plasmid p1AB5075 harbours the β-lactamase-encoding gene bla_GES-11. Class A β-lactamases like Bla_GES-11 use an active site serine residue to catalyse a ring-opening reaction of the β-lactam antibiotic. Due to the removal of the β-lactam ring, the key structural reactive centre of the antibiotic, the resulting product lacks antimicrobial activity. (b,c) Antibiotic disk diffusion assays. Resistant Acinetobacter strains were grown in the presence of cefotaxime, and culture supernatants were assessed by disk diffusion assay against the indicator organism M. luteus. Assays were performed in parallel with the donor strain A. baumannii AB5075-UW. (b) A. baumannii strains: A. baumannii AB048 transconjugant and A. baumannii AB053 transconjugant. (c) More genetically divergent Acinetobacter strains: A. nosocomialis DSM 102856 transconjugant, A. seifertii CIP 110471 transconjugant, A. haemolyticus ATCC 17906 transconjugant, and A. baylyi ATCC 33305 transconjugant.

Figure 5

Host strain diversity for T4SS-encoding plasmid member p1AB5075. Phylogenetic tree of recipient Acinetobacter strains for p1AB5075. Strain names are colour-coded based on the established clade [63] and subclade shown in Table 2, as reflected in the legend. Strain origin is indicated based on the symbols shown in the legend. The inset shown in light blue represents the A. baumannii lineage of Acinetobacter hosts.