Acinetobacter baumannii is a nosocomial pathogen that has emerged as a global threat because of high levels of resistance to many antibiotics, particularly those considered to be last-resort antibiotics, such as carbapenems. Although alterations in the efflux pump and outer membrane proteins can cause carbapenem resistance, the main mechanism is the acquisition of carbapenem-hydrolyzing oxacillinase-encoding genes. Of these, oxa23 is by far the most widespread in most countries, while oxa24 and oxa58 appear to be dominant in specific regions. Historically, much of the global spread of carbapenem resistance has been due to the dissemination of two major clones, known as global clones 1 and 2, although new lineages are now common in some parts of the world. The analysis of all publicly available genome sequences performed here indicates that ST2, ST1, ST79 and ST25 account for over 71 % of all genomes sequenced to date, with ST2 by far the most dominant type and oxa23 the most widespread carbapenem resistance determinant globally, regardless of clonal type. Whilst this highlights the global spread of ST1 and ST2, and the dominance of oxa23 in both clones, it could also be a result of preferential selection of carbapenem-resistant strains, which mainly belong to the two major clones. Furthermore, ~70 % of the sequenced strains have been isolated from five countries, namely the USA, PR China, Australia, Thailand and Pakistan, with only a limited number from other countries. These genomes are a vital resource, but it is currently difficult to draw an accurate global picture of this important superbug, highlighting the need for more comprehensive genome sequence data and genomic analysis.

Recent reports indicate the emergence of a new carbapenemase-producing Klebsiella pneumoniae clone, ST307. We sought to better understand the global epidemiology and evolution of this clone and evaluate its association with antimicrobial resistance (AMR) genes. We collated information from the literature and public databases and performed a comparative analysis of 95 ST307 genomes (including 37 that were newly sequenced). We show that ST307 emerged in the mid-1990s (nearly 20 years prior to its first report), is already globally distributed and is intimately associated with a conserved plasmid harbouring the bla_CTX-M-15 ESBL gene and several other AMR determinants. Our findings support the need for enhanced surveillance of this widespread ESBL clone in which carbapenem resistance has occasionally emerged.

The majority of Acinetobacter baumannii isolates that are multiply, extensively and pan antibiotic-resistant belong to two globally-disseminated clones, GC1 and GC2, that were first noticed in the 1970s. Here, we investigate microevolution and phylodynamics within GC1 via analysis of 45 whole genome sequences, including 25 sequenced for this study. The most recent common ancestor of GC1 arose around 1960, and later diverged into two phylogenetically distinct lineages. In the 1970s, the main lineage acquired the AbaR resistance island, conferring resistance to older antibiotics, via a horizontal gene transfer event. We estimate a mutation rate of ~5 single base substitutions per genome per year, and detected extensive recombination within GC1 genomes, introducing nucleotide diversity into the population at >20 times the substitution rate (r/m=22). The recombination events are non-randomly distributed in the genome and have created significant diversity within loci encoding outer surface molecules (including the capsular polysaccharide, the outer core of lipooligosaccharide and the outer membrane protein CarO), and spread antimicrobial resistance-conferring mutations affecting the gyrA and parC genes and IS insertions activating the ampC gene. Both GC1 lineages have accumulated resistance to newer antibiotics through various genetic mechanisms including the acquisition of plasmids and transposons or mutations in chromosomal genes. Our data show that GC1 has diversified into multiple successful extensively antibiotic resistant subclones that differ in their surface structures. This has important implications for all avenues of control including epidemiological tracking, antimicrobial therapy and vaccination.

Insertion sequences (IS) are small transposable elements, commonly found in bacterial genomes. Identifying the location of IS in bacterial genomes can be useful for a variety of purposes including epidemiological tracking and predicting antibiotic resistance. However IS are commonly present in multiple copies in a single genome, which complicates genome assembly and the identification of IS insertion sites. Here we present ISMapper, a mapping-based tool for identification of the site and orientation of IS insertions in bacterial genomes, directly from paired-end short read data. ISMapper was validated using three types of short read data: (i) simulated reads from a variety of species, (ii) Illumina reads from 5 isolates for which finished genome sequences were available for comparison, and (iii) Illumina reads from 7 Acinetobacter baumannii isolates for which predicted IS locations were tested using PCR. A total of 20 genomes, including 13 species and 32 distinct IS, were used for validation. ISMapper correctly identified 97 % of known IS insertions in the analysis of simulated reads, and 98 % in real Illumina reads. Subsampling of real Illumina reads to lower depths indicated ISMapper was able to correctly detect insertions for average genome-wide read depths >20x, although read depths >50x were required to obtain confident calls that were highly-supported by evidence from reads. All ISAba1 insertions identified by ISMapper in the A. baumannii genomes were confirmed by PCR. In each A. baumannii genome, ISMapper successfully identified an IS insertion upstream of the ampC beta-lactamase that could explain phenotypic resistance to third-generation cephalosporins. The utility of ISMapper was further demonstrated by profiling genome-wide IS6110 insertions in 138 publicly available Mycobacterium tuberculosis genomes, revealing lineage-specific insertions and multiple insertion hotspots. ISMapper provides a rapid and robust method for identifying IS insertion sites directly from short read data, with a high degree of accuracy demonstrated across a wide range of bacteria.

The misuse of antibiotics combined with a lack of newly developed ones is the main contributors to the current antibiotic resistance crisis. There is a dire need for new and alternative antibacterial options and nanotechnology could be a solution. Metal-based nanoparticles, particularly silver nanoparticles (NAg), have garnered widespread popularity due to their unique physicochemical properties and broad-spectrum antibacterial activity. Consequently, NAg has seen extensive incorporation in many types of products across the healthcare and consumer market. Despite clear evidence of the strong antibacterial efficacy of NAg, studies have raised concerns over the development of silver-resistant bacteria. Resistance to cationic silver (Ag⁺) has been recognised for many years, but it has recently been found that bacterial resistance to NAg is also possible. It is also understood that exposure of bacteria to toxic heavy metals like silver can induce the emergence of antibiotic resistance through the process of co-selection. Acinetobacter baumannii is a Gram-negative coccobacillus and opportunistic nosocomial bacterial pathogen. It was recently listed as the “number one” critical level priority pathogen because of the significant rise of antibiotic resistance in this species. NAg has proven bactericidal activity towards A. baumannii, even against strains that display multi-drug resistance. However, despite ample evidence of heavy metal (including silver; Ag⁺) resistance in this bacterium, combined with reports of heavy metal-driven co-selection of antibiotic resistance, little research has been dedicated to assessing the potential for NAg resistance development in A. baumannii. This is worrisome, as the increasingly indiscriminate use of NAg could promote the development of silver resistance in this species, like what has occurred with antibiotics.