On the last Wednesday of spring we were spoiled with the room on the top floor of the Australian Museum and magnificent views of Sydney, yes, once again! Joining us were not only our regular JAMS crowd, but also visitors from Europe (yes, that is really cold in Europe during this time of the year!)
A tale of two clones; multiple antibiotic resistance in Acinetobacter baumannii .
The importance of Acinetobacter baumannii in causing nosocomial infections was only recognised in the mid 1980s, making it the least studied of the bacteria that are now resistant to most of the antibiotics that are most important for treatment of such infections. We assembled a large collection of A. baumannii isolates recovered between 1999 and 2011 at hospitals in Sydney, Canberra, Brisbane, Melbourne, Newcastle and Adelaide. All but a few of those that are resistant to multiple antibiotics belong to one of two clonal groups that have recently been found to be globally distributed. Hence, these clones, global clone 1 and 2, have been in Australia at least since the mid 1990s.
Representatives of hospitals and of groups with different carbapenem and aminoglycoside resistance patterns in our collection have been sequenced together with the GC1 and GC2 reference strains from the early 1980s. Trees based on single nucleotide polymorphisms reveal significant diversity in the Australian isolates from one clone and little in the other. Most of the genes conferring resistance to older antibiotics are in the chromosome clustered in one island in GC1 and two in GC2 isolates. However, each of these islands is continually evolving, losing and gaining resistance genes. Further variation arises from the acquisition of different plasmids carrying further resistance genes. Other major but unexpected differences arising within the clones affect the exopolysaccharides. The capsule is an important virulence determinant, and substitution of large chromosomal segments leads to many distinct loci for capsule biosynthesis in each clone.
Does Acinetobacter baumannii have an O antigen?
Acinetobacter baumannii is amongst the most troublesome Gram-negative pathogens worldwide, due to strains that are resistant to multiple antibiotics, disinfection and periods of desiccation. Little is known about the virulence mechanisms, though a role for capsule has been demonstrated. Previous analysis of A. baumannii genome sequences identified a region of extensive diversity presumed to be involved in the synthesis of a surface polysaccharide, variously identified as O-antigen or capsule. We used bioinformatic tools to assess whether this polysaccharide is exported as capsule, or ligated to a lipid A-core oligosaccharide moiety to become the O antigen moiety of lipopolysaccharide. A gene for O-antigen ligase was not found, and we propose that A. baumannii strains produce a capsule (and lipid A-core oligosaccharide), but no lipopolysaccharide. 9 capsule types and 3 core types were found in the 10 completed genomes and more in draft genomes. Multiple capsule types were found in members of the 2 major clonal complexes, and this variation may contribute to the success of the A. baumannii clones by factoring in the evasion of the host immune response.
In September, JAMS was back into top gear, with a bigger audience, and a room with a view. Kent Lim from Macquarie University led off with a talk on his PhD work on the biocontrol agent Pseudomonas strain Pf5. As is often the case in science, things didn’t work out as expected, and Kent found that knocking out suspected pyochelin transporters led to an increase rather than a decrease in efflux of this siderophore and its metabolic precursors. Kent valiantly soldiered on, applying qRT-PCR and Biolog phenotype microarrays to untangle the problem, but unfortunately, this released even more worms from the seemingly-bottomless can provided by strain Pf5. It seems that these transporters may in fact also be regulatory proteins, explaining the unexpected pleiotropic effects of the knockouts.
Defining the effluxome of Acinetobacter baumannii
Acinetobacter baumannii is a Gram-negative opportunistic human pathogen known to cause a range of infections in hospitals. Despite their recent emergence, strains of A. baumannii, resistant to essentially all routinely used antibiotics, have been isolated from clinical settings. Bioinformatic analysis identified more than 50 transporter systems with a putative role in drug efflux in the genome of A. baumannii ATCC17978, representing ~2% of all its protein coding ORFs. Based on an assumption that drug transport is often associated with over-expression of a relevant efflux system in the presence of the substrate, high-throughput quantitative reverse-transcriptase PCR (qRT-PCR) has been performed after shock treatments with sub-inhibitory concentrations of antibiotics and differential expression of genes was assessed. This strategy has led to the discovery of novel drug efflux systems and defined physiological functions for previously characterised and novel pumps in drug resistance.
Efflux systems have evolved for millions of years before bacteria such as A. baumannii entered the hospital environment. Presumably, they have initially developed as mechanisms of resistance against naturally occurring substrates. To further characterize the role of efflux systems, cultures of A. baumannii were treated with bioactive natural compounds found in the environment, i.e. soil. These treatments resulted in significant changes in the transcription of efflux pumps indicating their possible role in the defence against compounds found in nature.
Increased expression of efflux systems was also observed when cells of A. baumannii were grown in biofilms compared to planktonic cultures which could suggest that efflux pumps may also play an important role in the functioning of these bacterial communities.
Transcriptome led microbial discovery.
Using a genome wide transcriptomic approach, Karl was able to unravel the role of the Pseudomonas global
activator system (GacA/GacS) in the regulation of an extremely broad range of functions including iron acquisition, oxidative stress response, secondary metabolism and motility. Similar work in Acinetobacter baumannii, a bacterium that is emerging as a major human pathogen due to multiple drug resistance, has revealed the antibiotic efflux to be major mode of resistance and led to the discovery of novel resistance proteins. Karl is a post-
doctoral fellow at Macquarie University working in Prof. Ian Paulsen’s group.