Sara Domingues

Natural Transformation Facilitates Transfer of Transposons, Integrons and Gene Cassettes between Bacterial Species

We have investigated to what extent natural transformation acting on free DNA substrates can facilitate transfer of mobile elements including transposons, integrons and/or gene cassettes between bacterial species. Naturally transformable cells of Acinetobacter baylyi were exposed to DNA from integron-carrying strains of the genera Acinetobacter, Citrobacter, Enterobacter, Escherichia, Pseudomonas, and Salmonella to determine the nature and frequency of transfer. Exposure to the various DNA sources resulted in acquisition of antibiotic resistance traits as well as entire integrons and transposons, over a 24 h exposure period. DNA incorporation was not solely dependent on integrase functions or the genetic relatedness between species. DNA sequence analyses revealed that several mechanisms facilitated stable integration in the recipient genome depending on the nature of the donor DNA; homologous or heterologous recombination and various types of transposition (Tn21-like and IS26-like). Both donor strains and transformed isolates were extensively characterized by antimicrobial susceptibility testing, integron- and cassette-specific PCRs, DNA sequencing, pulsed field gel electrophoreses (PFGE), Southern blot hybridizations, and by re-transformation assays. Two transformant strains were also genome-sequenced. Our data demonstrate that natural transformation facilitates interspecies transfer of genetic elements, suggesting that the transient presence of DNA in the cytoplasm may be sufficient for genomic integration to occur. Our study provides a plausible explanation for why sequence-conserved transposons, IS elements and integrons can be found disseminated among bacterial species. Moreover, natural transformation of integron harboring populations of competent bacteria revealed that interspecies exchange of gene cassettes can be highly efficient, and independent on genetic relatedness between donor and recipient. In conclusion, natural transformation provides a much broader capacity for horizontal acquisitions of genetic elements and hence, resistance traits from divergent species than previously assumed.

The blaIMP-5-carrying integron in a clinical Acinetobacter baumannii strain is flanked by miniature inverted-repeat transposable elements (MITEs)

Sir, IMP enzymes are one of the class B-type metallo-b-lactamases found in Acinetobacter baumannii. So far, nine IMP enzymes have been reported in this species, many as isolated cases (GenBank accession numbers DQ845788 and AB184977). IMP b-lactamases are often present in class 1 integrons, which, in turn, are embedded in transposons, resulting in highly transmissible genetic units. The majority of epidemiological investigations of the antimicrobial resistance encoded by class 1 integrons have depended on PCR primers that anneal to the conserved 5′CS and 3′CS ends of the integron. Analysis of PCR amplicons yields detailed insight into the presence and composition of resistance-encoding gene cassettes. However, cassette-targeted PCR analyses do not reveal the potential for and history of horizontal transmission of integrons. Novel insight into the horizontal transfer potential and mobility of integron-encoded resistance genes in pathogenic bacteria can be obtained from determination of the genetic regions surrounding the integrons. We have analysed the flanking sequences of the blaIMP-5-containing integron In76 in the clinical A. baumannii strain 65FFC to determine its unit of transfer. We report on the identification of a new integron mobilizable unit in this clinical bacterium. A. baumannii strain 65FFC is an imipenem-resistant clinical strain (MIC .32 mg/L) isolated from the urine of a man hospitalized in the neurotraumathology ward of the University Hospital of Coimbra, Portugal, in 1998. It produces the IMP-5 metallo-b-lactamase from the blaIMP-5 gene cassette present in a class 1 integron (GenBank accession number AF290912). The strain is resistant to all b-lactam antibiotics (except ampicillin/sulbactam). The flanking regions of In76 as well as the integron sequence (10216 bp) were determined by direct genomic DNA sequencing. Upand downstream primer walking was based on initial annealing of the sequencing primer in conserved parts of the integron (GenBank accession number M73819). Sequencing was performed using BigDye chemistry (Applied Biosystems) and the sequences were edited and aligned in Sequencher v.4.2.2 (GeneCodes, Ann Arbor, MI, USA), and identified using BLASTN (www.ncbi.nlm.nih.gov). A total of 3258 bp of sequence was determined upstream of the 5′CS region and 3564 bp downstream of the 3′CS region. In76 was found embedded in a Tn402-like transposon. A miniature inverted-repeat transposable element (MITE)-like structure of 439 bp was identified immediately adjacent to the beginning of the 5′CS of the integron, preceded by an incomplete putative transposase. Downstream of the 3′CS, two genes (tniBD1 and tniA) were identified that belong to a common defective transposition module of integrons. However, the tniA gene was interrupted by a second MITE with an identical 439 bp sequence to the one at the 5′CS flanking region. An interrupted putative transposase followed the MITE structure further downstream (Figure 1a). The nucleotide sequence obtained in this study has been deposited in the GenBank database (accession number JF810083). The presence of the complete In76 integron and the two immediately flanking MITEs, together with the identification of a 5 bp target site duplication adjacent to both the MITEs, strongly suggests that the entire structure had inserted into the transposase gene through transposition. Bioinformatic analysis also revealed that another 439 bp MITE structure, 100% identical to our isolate from Portugal, was previously found flanking a class 1 integron of an Acinetobacter johnsonii isolated from the digestive tract of an ocean prawn in Australia (GenBank accession number FJ711439). Although the MITE-like structures are inserted in the same relative position within the integron, they flank two different class 1 integrons with unrelated gene cassettes, and the entire structure is inserted in a different genetic context (Figure 1a and b). The acquisition of the integron by A. johnsonii by a MITE-facilitated transposition-like mechanism was also proposed by Gillings et al. The 100% identical MITE-like structure present in two different Acinetobacter species from different continents suggests that MITEs can disseminate horizontally and act as mobilizable vectors for resistance dissemination. MITEs are non-autonomous mobile elements consisting of small repeat sequences, which do not encode proteins and are found randomly inserted in the genome of diverse bacteria. The mobilization of MITE-like structures by transposition has

Global dissemination patterns of common gene cassette arrays in class 1 integrons.

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 >300 cassette arrays have been described, < 10 array compositions prevail in the reports related to 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 amongst bacterial populations and species. Clonal expansion also contributes to the current prevalence 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.

Identical Miniature Inverted Repeat Transposable Elements Flank Class 1 Integrons in Clinical Isolates of Acinetobacter spp.

ABSTRACT Miniature inverted repeat transposable elements (MITEs) have been identified flanking class 1 integrons. We have identified and characterized a 439-bp MITE-like structure in seven Acinetobacter species isolates from Portugal and Brazil. The complete sequence similarity of the elements and flanking regions suggests that MITEs may act as mobilizable vectors for the dissemination of integrons.

Insights on the Horizontal Gene Transfer of Carbapenemase Determinants in the Opportunistic Pathogen Acinetobacter baumannii

Horizontal gene transfer (HGT) is a driving force to the evolution of bacteria. The fast emergence of antimicrobial resistance reflects the ability of genetic adaptation of pathogens. Acinetobacter baumannii has emerged in the last few decades as an important opportunistic nosocomial pathogen, in part due to its high capacity of acquiring resistance to diverse antibiotic families, including to the so-called last line drugs such as carbapenems. The rampant selective pressure and genetic exchange of resistance genes hinder the effective treatment of resistant infections. A. baumannii uses all the resistance mechanisms to survive against carbapenems but production of carbapenemases are the major mechanism, which may act in synergy with others. A. baumannii appears to use all the mechanisms of gene dissemination. Beyond conjugation, the mostly reported recent studies point to natural transformation, transduction and outer membrane vesicles-mediated transfer as mechanisms that may play a role in carbapenemase determinants spread. Understanding the genetic mobilization of carbapenemase genes is paramount in preventing their dissemination. Here we review the carbapenemases found in A. baumannii and present an overview of the current knowledge of contributions of the various HGT mechanisms to the molecular epidemiology of carbapenem resistance in this relevant opportunistic pathogen.

Various pathways leading to the acquisition of antibiotic resistance by natural transformation

Natural transformation can lead to exchange of DNA between taxonomically diverse bacteria. In the case of chromosomal DNA, homology-based recombination with the recipient genome is usually necessary for heritable stability. In our recent study, we have shown that natural transformation can promote the transfer of transposons, IS elements, and integrons and gene cassettes, largely independent of the genetic relationship between the donor and recipient bacteria. Additional results from our study suggest that natural transformation with species-foreign DNA might result in the uptake of a wide range of DNA fragments; leading to changes in the antimicrobial susceptibility profile and contributing to the generation of antimicrobial resistance in bacteria.

Membrane vesicles and horizontal gene transfer in prokaryotes

Membrane vesicles (MVs) are released from all living cells. MVs are lumen-containing spheres of lipid-bilayers derived from the cell surface. MVs are biologically active and contain various components, including genetic material. Both chromosomal and plasmid DNA, as well as different types of RNA have been detected in MVs. Vesicle-mediated transfer of genes coding for antibiotic resistance, virulence and metabolic traits has been reported in Gram-negative and Gram-positive bacteria and in Archaea. MVs can persist over time in natural environments. Here we review the characteristics of and the role of MVs in horizontal gene transfer (HGT) processes in prokaryotes.

Involvement of aph(3′)-IIa in the formation of mosaic aminoglycoside resistance genes in natural environments

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 three 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.

Characterization of a novel international clonal complex (CC32) of Acinetobacter baumannii with epidemic potential

Twelve non-replicate Acinetobacter baumannii isolates from five European hospitals, Kuwait, and the US military healthcare system collected between 1980 and 2005 revealed a new clone, CC32. These included representative isolates of outbreaks/cross-infections. Antimicrobial susceptibility and carbapenem-resistant genetic traits varied. The widespread occurrence, the association with an outbreak and the carbapenem resistance indicate that CC32 has epidemic potential.

Interplay between Colistin Resistance, Virulence and Fitness in Acinetobacter baumannii

Acinetobacter baumannii is an important opportunistic nosocomial pathogen often resistant to multiple antibiotics classes. Colistin, an “old” antibiotic, is now considered a last-line treatment option for extremely resistant isolates. In the meantime, resistance to colistin has been reported in clinical A. baumannii strains. Colistin is a cationic peptide that disrupts the outer membrane (OM) of Gram-negative bacteria. Colistin resistance is primarily due to post-translational modification or loss of the lipopolysaccharide (LPS) molecules inserted into the outer leaflet of the OM. LPS modification prevents the binding of polymyxin to the bacterial surface and may lead to alterations in bacterial virulence. Antimicrobial pressure drives the evolution of antimicrobial resistance and resistance is often associated with a reduced bacterial fitness. Therefore, the alterations in LPS may induce changes in the fitness of A. baumannii. However, compensatory mutations in clinical A. baumannii may ameliorate the cost of resistance and may play an important role in the dissemination of colistin-resistant A. baumannii isolates. The focus of this review is to summarize the colistin resistance mechanisms, and understand their impact on the fitness and virulence of bacteria and on the dissemination of colistin-resistant A. baumannii strains.