Kaare Magne Nielsen

Gram-negative bacteria are intrinsically resistant to many commercialized antibiotics. The outer membrane (OM) of Gram-negative bacteria prevents the entry of such antibiotics. Outer membrane vesicles (OMV) are naturally released from the OM of Gram-negative bacteria for a range of purposes, including competition with other bacteria. OMV may carry, as part of the membrane or lumen, molecules with antibacterial activity. Such OMV can be exposed to and can fuse with the cell surface of different bacterial species. In this review we consider how OMV can be used as tools to deliver antimicrobial agents. This includes the characteristics of OMV production and how this process can be used to create the desired antibacterial activity of OMV.

The role of vesicle-mediated gene transfer in Acinetobacter baumannii populations has been investigated in the last decade. Importantly, outer membrane vesicles (OMVs) secreted from A. baumannii cells have proven to be efficient agents of transfer of antimicrobial resistance genes to other bacterial species. However, the measurement of vesicle-mediated transfer depends on many experimental parameters. Here, we describe an experimental method useful to study transfer of DNA via membrane vesicles of A. baumannii in various bacterial populations.

Horizontal gene transfer events provide the basis for extensive dissemination of antimicrobial resistance traits between bacterial populations. Conjugation is considered to be the most frequent mechanism behind new resistance acquisitions in clinical pathogens but does not fully explain the resistance patterns seen in some bacterial genera. Gene transfer by natural transformation has been described for numerous clinical isolates, including some Acinetobacter species. The main aim of this study was to determine to what extent clinical, resistant Acinetobacter spp. isolates, express competence for natural transformation. Twenty-two clinical Acinetobacter spp. isolates collected over a 16-year time period, from five different geographical separated and/or distinct Portuguese Hospitals were tested for natural transformability. Fourteen isolates, including 11 A. baumannii, 2 A. nosocomialis and 1 Acinetobacter sp., were identified as competent on semisolid media facilitating surface-motility. Competent Acinetobacter isolates were found in all the hospitals tested. Furthermore, osmolarity was shown to influence the uptake of exogenous DNA by competent A. baumannii A118. Our study demonstrates that natural competence is common among clinical isolates of Acinetobacter spp., and hence likely an important trait for resistance acquisition.

In a screen for unexplained mutation events we identified a previously unrecognized mechanism generating clustered DNA polymorphisms such as microindels and cumulative SNPs. The mechanism, short-patch double illegitimate recombination (SPDIR), facilitates short single-stranded DNA molecules to invade and replace genomic DNA through two joint illegitimate recombination events. SPDIR is controlled by key components of the cellular genome maintenance machinery in the gram-negative bacterium Acinetobacter baylyi. The source DNA is primarily intragenomic but can also be acquired through horizontal gene transfer. The DNA replacements are nonreciprocal and locus independent. Bioinformatic approaches reveal occurrence of SPDIR events in the gram-positive human pathogen Streptococcus pneumoniae and in the human genome. Significance Clustered genomic polymorphisms in DNA, such as microindels and stretches of nucleotide changes, play an important role in genome evolution. Here, we report a mutation mechanism responsible for such genomic polymorphisms where short, single-stranded DNA molecules invade double-stranded DNA and replace short genomic segments. We show, in a bacterial model organism, that the genomic replacements occur with very low levels of sequence identity (microhomologies). The invading DNA can be of intagenomic or foreign origin. Genotoxic stress, horizontally taken-up DNA, or lack of genome maintenance functions increase the mutation frequency up to 7,000-fold. Bioinformatic approaches suggest that this class of mutations is widespread in prokaryotes and eukaryotes and may have a role in tumorigenesis.

Integrons are genetic elements that contain a site-specific recombination system able to capture, express and exchange gene cassettes. Mobile integrons are widespread and often confer resistance to multiple antibiotics, due to the expression of the arrays of gene cassettes they carry. Although more than 300 cassette arrays have been described, less than 10 array compositions prevail in the reports related with class 1 integrons. These common arrays are found in a broad variety of hosts and environments, highlighting the high level of horizontal dissemination of these elements among bacterial populations and species. Clonal expansion also contributes to the current prevalence of and inter-regional spread of integron-carrying bacterial species. Here we review the dissemination pattern of common cassette arrays with a focus on the bacterial species, the geographical dispersal pattern and the environments in which they reside. Conserved arrays of gene cassettes are found in at least 74 countries and 72 species present in different environments. The factors governing the further spread and population dynamics of these cassette arrays remain to be determined.

Intragenic recombination leading to mosaic gene formation is known to alter resistance profiles for particular genes and bacterial species. Few studies have examined to what extent aminoglycoside resistance genes undergo intragenic recombination. We screened the GenBank database for mosaic gene formation in homologs of the aph(3’)-IIa (nptII) gene. APH(3’)-IIa inactivates important aminoglycoside antibiotics. The gene is widely used as a selectable marker in biotechnology and enters the environment via laboratory discharges and the release of transgenic organisms. Such releases may provide opportunities for recombination in competent environmental bacteria. The retrieved GenBank sequences were grouped in 3 datasets comprising river water samples, duck pathogens and full-length variants from various bacterial genomes and plasmids. Analysis for recombination in these datasets was performed with the Recombination Detection Program, RDP4, and the Genetic Algorithm for Recombination Detection, GARD. From a total of 89 homologous sequences, 83% showed 99% - 100% sequence identity with aph(3’)-IIa originally described as part of transposon Tn5. Fifty one were unique sequence variants eligible for recombination analysis. Only a single recombination event was identified with high confidence and indicated the involvement of aph(3’)-IIa in the formation of a mosaic gene located on a plasmid of environmental origin in the multi-resistant isolate Pseudomonas aeruginosa PA96. The available data suggest that aph(3’)-IIa is not an archetypical mosaic gene as the divergence between the described sequence variants and the number of detectable recombination events is low. This is in contrast to the numerous mosaic alleles reported for certain penicillin or tetracycline resistance determinants.