Plasmid-encoded efflux pumps; empowering Acinetobacter in the battle against antimicrobial resistance
Posted on June 26, 2023 by Dr Varsha Naidu and Associate Professor Karl Hassan
Dr Varsha Naidu and Associate Professor Karl Hassan take us behind the scenes of their latest publication 'AadT, a new weapon in Acinetobacter’s fight against antibiotics 'published in Microbiology.
Antimicrobial resistance has been declared by the WHO as one of the most significant global health crises facing humanity. ESKAPE pathogens, a term coined by the Infectious Disease Society of America, encompass the notorious group of pathogens that reign as the primary instigators of hospital-acquired infections worldwide, posing a significant risk to public health. These pathogens exhibit an astonishing and unsettling aptitude for evading treatments. In light of its remarkable ability to evade existing treatment options, carbapenem-resistant Acinetobacter baumannii has been identified by the WHO as one of the top three bacterial groups requiring immediate attention for the development of novel antibiotics.
In A. baumannii, multidrug efflux pumps play a crucial role in antimicrobial resistance, as a single pump can confer resistance to a broad-spectrum of antimicrobials. A significant number of these pumps are found in the core genomes of the species, indicating vertical inheritance. Others demonstrate associations with mobile genetic elements, such as plasmids and transposons suggesting horizontal transfer between strains. Efflux pumps, particularly those belonging to the Resistance/Nodulation/Division (RND) superfamily, have been associated with significant levels of antimicrobial resistance. Within A. baumannii, three members of this family, namely AdeAB(C), AdeFGH, and AdeIJK, have been extensively studied and characterised. These pumps are encoded in the chromosome of A. baumannii and can be considered a core feature of the species.
In recent work, we and other groups surveyed the genomes of diverse strains within the Acinetobacter genus to identify homologs of these major multidrug efflux pumps and infer their levels of conservation across the genus. We found that AdeIJK was highly conserved across the genus and a core feature that has likely been retained since the inception of the genus. AdeFGH was well conserved within sub-lineages of Acinetobacter, such as the A. calcoaceticus/baumannii (ACB) group and clade containing A. haemolyticus, whereas homologs were rarely observed outside of these lineages. Contrarily, the distribution of AdeAB(C) was the most variable. It was found within most strains that carry AdeFGH, but its loci varied among different strains. Certain strains harboured multiple putative homologs of the AdeAB(C) system. Examination of these strains showed that they carried a copy of the genes encoding AdeAB(C) on a plasmid.
Plasmid encoded RND family pumps are found in some species, but rarer than those carried chromosomally. Therefore, we investigated the plasmids encoding efflux pumps homologous to AdeAB(C). Previous reports highlighted that these plasmids may contain a composite like transposon that carries numerous resistance genes, including those conferring resistance to the last line antibiotic carbapenem. Of particular interest to us was the frequent presence of a gene encoding a putative efflux pump from the Major Facilitator Superfamily (MFS) downstream adeAB(C) homologs in many Acinetobacter plasmids. At least 300 strains across 23 designated Acinetobacter species and Acinetobacter strains lacking species designation were found to carry genes encoding homologs of this MFS efflux pump. A detailed examination of the genetic context of these genes found that they were either located downstream of the IS18/adeRS/adeABC cluster, or they were located between homologs of adeAB and adeRS gene pairs, while adeC and the IS18 transposase gene were absent. The absence of adeC from adeAB loci in Acinetobacter genomes is not uncommon, since AdeAB can function with AdeK from the AdeIJK system as an alternative outer membrane protein.
To investigate the relationships of the MFS efflux pump relative to other members of the MFS, a phylogenetic analysis was performed. This analysis found that the pump was a member of the MFS sub-family called the Drug:H+ antiporter 2 (DHA2), whose members frequently contribute to biocide tolerance in a range of both Gram-positive and Gram-negative pathogens. The close proximity of genes encoding this MFS efflux pump to adeAB(C) genes, the classification of the pump within the DHA2 family, indicated that it may also contribute to antimicrobial resistance. We assessed its capacity to confer resistance to eight diverse antimicrobials, including antibiotics, dyes and biocides, and compared its resistance profile to the related multidrug efflux pump AmvA. Cells expressing the MFS pump were less susceptible to all eight tested antimicrobials, and surpassed AmvA in conferring higher tolerance levels to tetracycline, DAPI, ethanol, and ethidium bromide. These findings indicate that this efflux pump, similar to AmvA, has the ability to recognise a wide range of antimicrobials, establishing it as a novel multidrug transport protein in Acinetobacter. We called the efflux pump AadT, as the Acinetobacter antimicrobial drug transporter.
The proximity of aadT to adeAB(C) and adeRS genes suggests that the expression of aadT genes may be regulated by AdeRS and possibly co-expressed alongside adeAB(C). The discovery of genes encoding novel multidrug efflux pumps as well as new variants of AdeAB(C) and AdeRS in plasmids of Acinetobacter is significant. It remains uncertain whether plasmid encoded AdeAB(C) exhibits distinct substrate preferences compared to those encoded chromosomally. Understanding such details is of utmost importance in comprehending the antibiotic resistance capabilities of pathogenic Acinetobacter and the future health threat of plasmids carrying these genes.