Genomic analysis of CTX-M-115 and OXA-23/-72 co-producing Acinetobacter baumannii, and their potential to spread resistance genes by natural transformation.

OBJECTIVES: To characterize Acinetobacter baumannii strains co-producing the ESBL CTX-M-115 and carbapenem-hydrolysing class D ß-lactamases (CHDLs), and to assess the potential diffusion of their resistance genes by horizontal transfer. METHODS: Nineteen CTX-M-115/CHDL-positive A. baumannii were collected between 2015 and 2019 from patients hospitalized in France. Their whole-genome sequences were determined on Illumina and Oxford Nanopore platforms and were compared through core-genome MLST (cgMLST) and SNP analyses. Transferability of resistance genes was investigated by natural transformation assays. RESULTS: Eighteen strains were found to harbour CHDL OXA-72, and another one CHDL OXA-23, in addition to CTX-M-115, narrow-spectrum ß-lactamases and aminoglycoside resistance determinants including ArmA. cgMLST typing, as well as Oxford Scheme ST and K locus typing, confirmed that 17 out of the 18 CTX-M-115/OXA-72 isolates belonged to new subclades within clonal complex 78 (CC78). The chromosomal region carrying the blaCTX-M-115 gene appeared to vary greatly both in gene content and in length (from 20 to 79 kb) among the strains, likely because of IS26-mediated DNA rearrangements. The blaOXA-72 gene was localized on closely related plasmids showing structural variations that occurred between pdif sites. Transfer of all the ß-lactamase genes, as well as aminoglycoside resistance determinants to a drug-susceptible A. baumannii recipient, was easily obtained in vitro by natural transformation. CONCLUSIONS: This work highlights the propensity of CC78 isolates to collect multiple antibiotic resistance genes, to rearrange and to pass them to other A. baumannii strains via natural transformation. This process, along with mobile genetic elements, likely contributes to the considerable genomic plasticity of clinical strains, and to the diversity of molecular mechanisms sustaining their multidrug resistance.

Interbacterial Transfer of Carbapenem Resistance and Large Antibiotic Resistance Islands by Natural Transformation in Pathogenic Acinetobacter.

Acinetobacter baumannii infection poses a major health threat, with recurrent treatment failure due to antibiotic resistance, notably to carbapenems. While genomic analyses of clinical strains indicate that homologous recombination plays a major role in the acquisition of antibiotic resistance genes, the underlying mechanisms of horizontal gene transfer often remain speculative. Our understanding of the acquisition of antibiotic resistance is hampered by the lack of experimental systems able to reproduce genomic observations. We here report the detection of recombination events occurring spontaneously in mixed bacterial populations and which can result in the acquisition of resistance to carbapenems. We show that natural transformation is the main driver of intrastrain but also interstrain recombination events between A. baumannii clinical isolates and pathogenic species of Acinetobacter. We observed that interbacterial natural transformation in mixed populations is more efficient at promoting the acquisition of large resistance islands (AbaR4 and AbaR1) than when the same bacteria are supplied with large amounts of purified genomic DNA. Importantly, analysis of the genomes of the recombinant progeny revealed large recombination tracts (from 13 to 123 kb) similar to those observed in the genomes of clinical isolates. Moreover, we highlight that transforming DNA availability is a key determinant of the rate of recombinants and results from both spontaneous release and interbacterial predatory behavior. In the light of our results, natural transformation should be considered a leading mechanism of genome recombination and horizontal gene transfer of antibiotic resistance genes in Acinetobacter baumannii. IMPORTANCE Acinetobacter baumannii is a multidrug-resistant pathogen responsible for difficult-to-treat hospital-acquired infections. Understanding the mechanisms leading to the emergence of the multidrug resistance in this pathogen today is crucial. Horizontal gene transfer is assumed to largely contribute to this multidrug resistance. However, in A. baumannii, the mechanisms leading to genome recombination and the horizontal transfer of resistance genes are poorly understood. We describe experimental evidence that natural transformation, a horizontal gene transfer mechanism recently highlighted in A. baumannii, allows the highly efficient interbacterial transfer of genetic elements carrying resistance to last-line antibiotic carbapenems. Importantly, we demonstrated that natural transformation, occurring in mixed populations of Acinetobacter, enables the transfer of large resistance island-mobilizing multiple-resistance genes.

Environmental Free-Living Amoebae Can Predate on Diverse Antibiotic-Resistant Human Pathogens.

Here, we sought to test the resistance of human pathogens to unaltered environmental free-living amoebae. Amoebae are ubiquitous eukaryotic microorganisms and important predators of bacteria. Environmental amoebae have also been proposed to serve as both potential reservoirs and training grounds for human pathogens. However, studies addressing their relationships with human pathogens often rely on a few domesticated amoebae that have been selected to feed on rich medium, thereby possibly overestimating the resistance of pathogens to these predatory phagocytes. From an open-air composting site, we recovered over 100 diverse amoebae that were able to feed on Acinetobacter baumannii and Klebsiella pneumoniae. In a standardized and quantitative assay for predation, the isolated amoebae showed a broad predation spectrum, killing clinical isolates of A. baumannii, K. pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. Interestingly, A. baumannii, which was previously reported to resist predation by laboratory strains of Acanthamoeba, was efficiently consumed by closely related environmental amoebae. The isolated amoebae were capable of feeding on highly virulent carbapenem-resistant or methicillin-resistant clinical isolates. In conclusion, the natural environment is a rich source of amoebae with broad-spectrum bactericidal activities, including against antibiotic-resistant isolates. IMPORTANCE Free-living amoebae have been proposed to play an important role in hosting and disseminating various human pathogens. The resistance of human pathogens to predation by amoebae is often derived from in vitro experiments using model amoebae. Here, we sought to isolate environmental amoebae and to test their predation on diverse human pathogens, with results that challenge conclusions based on model amoebae. We found that the natural environment is a rich source of diverse amoebae with broad-spectrum predatory activities against human pathogens, including highly virulent and antibiotic-resistant clinical isolates.

Scarless Removal of Large Resistance Island AbaR Results in Antibiotic Susceptibility and Increased Natural Transformability in Acinetobacter baumannii.

With a great diversity in gene composition, including multiple putative antibiotic resistance genes, AbaR islands are potential contributors to multidrug resistance in Acinetobacter baumannii However, the effective contribution of AbaR to antibiotic resistance and bacterial physiology remains elusive. To address this, we sought to accurately remove AbaR islands and restore the integrity of their insertion site. To this end, we devised a versatile scarless genome editing strategy. We performed this genetic modification in two recent A. baumannii clinical strains: the strain AB5075 and the nosocomial strain AYE, which carry AbaR11 and AbaR1 islands of 19.7 kbp and 86.2 kbp, respectively. Antibiotic susceptibilities were then compared between the parental strains and their AbaR-cured derivatives. As anticipated by the predicted function of the open reading frame (ORF) of this island, the antibiotic resistance profiles were identical between the wild type and the AbaR11-cured AB5075 strains. In contrast, AbaR1 carries 25 ORFs, with predicted resistance to several classes of antibiotics, and the AYE AbaR1-cured derivative showed restored susceptibility to multiple classes of antibiotics. Moreover, curing of AbaRs restored high levels of natural transformability. Indeed, most AbaR islands are inserted into the comM gene involved in natural transformation. Our data indicate that AbaR insertion effectively inactivates comM and that the restored comM is functional. Curing of AbaR consistently resulted in highly transformable and therefore easily genetically tractable strains. Emendation of AbaR provides insight into the functional consequences of AbaR acquisition.

Bacterial Transformation Buffers Environmental Fluctuations through the Reversible Integration of Mobile Genetic Elements.

Horizontal gene transfer (HGT) promotes the spread of genes within bacterial communities. Among the HGT mechanisms, natural transformation stands out as being encoded by the bacterial core genome. Natural transformation is often viewed as a way to acquire new genes and to generate genetic mixing within bacterial populations. Another recently proposed function is the curing of bacterial genomes of their infectious parasitic mobile genetic elements (MGEs). Here, we propose that these seemingly opposing theoretical points of view can be unified. Although costly for bacterial cells, MGEs can carry functions that are at points in time beneficial to bacteria under stressful conditions (e.g., antibiotic resistance genes). Using computational modeling, we show that, in stochastic environments, an intermediate transformation rate maximizes bacterial fitness by allowing the reversible integration of MGEs carrying resistance genes, although these MGEs are costly for host cell replication. Based on this dual function (MGE acquisition and removal), transformation would be a key mechanism for stabilizing the bacterial genome in the long term, and this would explain its striking conservation.IMPORTANCE Natural transformation is the acquisition, controlled by bacteria, of extracellular DNA and is one of the most common mechanisms of horizontal gene transfer, promoting the spread of resistance genes. However, its evolutionary function remains elusive, and two main roles have been proposed: (i) the new gene acquisition and genetic mixing within bacterial populations and (ii) the removal of infectious parasitic mobile genetic elements (MGEs). While the first one promotes genetic diversification, the other one promotes the removal of foreign DNA and thus genome stability, making these two functions apparently antagonistic. Using a computational model, we show that intermediate transformation rates, commonly observed in bacteria, allow the acquisition then removal of MGEs. The transient acquisition of costly MGEs with resistance genes maximizes bacterial fitness in environments with stochastic stress exposure. Thus, transformation would ensure both a strong dynamic of the bacterial genome in the short term and its long-term stabilization.

Fluorescence-Based Detection of Natural Transformation in Drug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a nosocomial agent with a high propensity for developing resistance to antibiotics. This ability relies on horizontal gene transfer mechanisms occurring in the Acinetobacter genus, including natural transformation. To study natural transformation in bacteria, the most prevalent method uses selection for the acquisition of an antibiotic resistance marker in a target chromosomal locus by the recipient cell. Most clinical isolates of A. baumannii are resistant to multiple antibiotics, limiting the use of such selection-based methods. Here, we report the development of a phenotypic and selection-free method based on flow cytometry to detect transformation events in multidrug-resistant (MDR) clinical A. baumannii isolates. To this end, we engineered a translational fusion between the abundant and conserved A. baumannii nucleoprotein (HU) and the superfolder green fluorescent protein (sfGFP). The new method was benchmarked against the conventional antibiotic selection-based method. Using this new method, we investigated several parameters affecting transformation efficiencies and identified conditions of transformability one hundred times higher than those previously reported. Using optimized transformation conditions, we probed natural transformation in a set of MDR clinical and nonclinical animal A. baumannii isolates. Regardless of their origin, the majority of the isolates displayed natural transformability, indicative of a conserved trait in the species. Overall, this new method and optimized protocol will greatly facilitate the study of natural transformation in the opportunistic pathogen A. baumanniiIMPORTANCE Antibiotic resistance is a pressing global health concern with the rise of multiple and panresistant pathogens. The rapid and unfailing resistance to multiple antibiotics of the nosocomial agent Acinetobacter baumannii, notably to carbapenems, prompt to understand the mechanisms behind acquisition of new antibiotic resistance genes. Natural transformation, one of the horizontal gene transfer mechanisms in bacteria, was only recently described in A. baumannii and could explain its ability to acquire resistance genes. We developed a reliable method to probe and study natural transformation mechanism in A. baumannii More broadly, this new method based on flow cytometry will allow experimental detection and quantification of horizontal gene transfer events in multidrug-resistant A. baumannii.

Expanding the Kinome World: A New Protein Kinase Family Widely Conserved in Bacteria.

Fine tuning of signaling pathways is essential for cells to cope with sudden environmental variations. This delicate balance is maintained in particular by protein kinases that control the activity of target proteins by reversible phosphorylation. In addition to homologous eukaryotic enzymes, bacteria have evolved some specific Ser/Thr/Tyr protein kinases without any structural resemblance to their eukaryotic counterparts. Here, we show that a previously identified family of ATPases, broadly conserved among bacteria, is in fact a new family of protein kinases with a Ser/Thr/Tyr kinase activity. A prototypic member of this family, YdiB from Bacillus subtilis, is able to autophosphorylate and to phosphorylate a surrogate substrate, the myelin basic protein. Two crystal structures of YdiB were solved (1.8 and 2.0Å) that display a unique ATP-binding fold unrelated to known protein kinases, although a conserved HxD motif is reminiscent of that found in Hanks-type protein kinases. The effect of mutations of conserved residues further highlights the unique nature of this new protein kinase family that we name ubiquitous bacterial kinase. We investigated the cellular role of YdiB and showed that a ∆ydiB mutant was more sensitive to paraquat treatment than the wild type, with ~13% of cells with an aberrant morphology. In addition, YdiE, which is known to participate with both YdiC and YdiB in an essential chemical modification of some specific tRNAs, is phosphorylated in vitro by YdiB. These results expand the boundaries of the bacterial kinome and support the involvement of YdiB in protein translation and resistance to oxidative stress in B. subtilis.

Detection and genetic characterization of Seoul virus from commensal brown rats in France.

BACKGROUND: Hantaviruses are single-stranded RNA viruses, which are transmitted to humans primarily via inhalation of aerosolised virus in contaminated rodent urine and faeces. Whilst infected reservoir hosts are asymptomatic, human infections can lead to two clinical manifestations, haemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), with varying degrees of clinical severity. The incidence of rodent and human cases of Seoul virus (SEOV) in Europe has been considered to be low, and speculated to be driven by the sporadic introduction of infected brown rats (Rattus norvegicus) via ports. METHODS: Between October 2010 and March 2012, 128 brown rats were caught at sites across the Lyon region in France. RESULTS: SEOV RNA was detected in the lungs of 14% (95% CI 8.01-20.11) of brown rats tested using a nested pan-hantavirus RT-PCR (polymerase gene). Phylogenetic analysis supports the inclusion of the Lyon SEOV within Lineage 7 with SEOV strains originating from SE Asia and the previously reported French & Belgian SEOV strains. Sequence data obtained from the recent human SEOV case (Replonges) was most similar to that obtained from one brown rat trapped in a public park in Lyon city centre. We obtained significantly improved recovery of virus genome sequence directly from SEOV infected lung material using a simple viral enrichment approach and NGS technology. CONCLUSIONS: The detection of SEOV in two wild caught brown rats in the UK and the multiple detection of SEOV infected brown rats in the Lyon region of France, suggests that SEOV is circulating in European brown rats. Under-reporting and difficulties in identifying the hantaviruses associated with HFRS may mask the public health impact of SEOV in Europe.

O-Acetylation of peptidoglycan is required for proper cell separation and S-layer anchoring in Bacillus anthracis.

O-Acetylation of the MurNAc moiety of peptidoglycan is typically associated with bacterial resistance to lysozyme, a muramidase that serves as a central component of innate immunity. Here, we report that the peptidoglycan of Bacillus anthracis, the etiological agent of anthrax, is O-acetylated and that, unusually, this modification is produced by two unrelated families of O-acetyltransferases. Also, in contrast to other bacteria, O-acetylation of B. anthracis peptidoglycan is combined with N-deacetylation to confer resistance of cells to lysozyme. Activity of the Pat O-acetyltransferases is required for the separation of the daughter cells following bacterial division and for anchoring of one of the major S-layer proteins. Our results indicate that peptidoglycan O-acetylation modulates endogenous muramidase activity affecting the cell-surface properties and morphology of this important pathogen.

A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments.

Bacteria can respond to adverse environmental conditions by drastically reducing or even ceasing metabolic activity. They must then determine that conditions have improved before exiting dormancy, and one indication of such a change is the growth of other bacteria in the local environment. Growing bacteria release muropeptide fragments of the cell wall into the extracellular milieu, and we report here that these muropeptides are potent germinants of dormant Bacillus subtilis spores. The ability of a muropeptide to act as a germinant is determined by the identity of a single amino acid. A well-conserved, eukaryotic-like Ser/Thr membrane kinase containing an extracellular domain capable of binding peptidoglycan is necessary for this response, and a small molecule that stimulates related eukaryotic kinases is sufficient to induce germination. Another small molecule, staurosporine, that inhibits related eukaryotic kinases blocks muropeptide-dependent germination. Thus, in contrast to traditional antimicrobials that inhibit metabolically active cells, staurosporine acts by blocking germination of dormant spores.

Role of spore coat proteins in the resistance of Bacillus subtilis spores to Caenorhabditis elegans predation.

Bacterial spores are resistant to a wide range of chemical and physical insults that are normally lethal for the vegetative form of the bacterium. While the integrity of the protein coat of the spore is crucial for spore survival in vitro, far less is known about how the coat provides protection in vivo against predation by ecologically relevant hosts. In particular, assays had characterized the in vitro resistance of spores to peptidoglycan-hydrolyzing enzymes like lysozyme that are also important effectors of innate immunity in a wide variety of hosts. Here, we use the bacteriovorous nematode Caenorhabditis elegans, a likely predator of Bacillus spores in the wild, to characterize the role of the spore coat in an ecologically relevant spore-host interaction. We found that ingested wild-type Bacillus subtilis spores were resistant to worm digestion, whereas vegetative forms of the bacterium were efficiently digested by the nematode. Using B. subtilis strains carrying mutations in spore coat genes, we observed a correlation between the degree of alteration of the spore coat assembly and the susceptibility to the worm degradation. Surprisingly, we found that the spores that were resistant to lysozyme in vitro can be sensitive to C. elegans digestion depending on the extent of the spore coat structure modifications.

Concert of regulators to switch on LEE expression in enterohemorrhagic Escherichia coli O157:H7: interplay between Ler, GrlA, HNS and RpoS.

Enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains carry a pathogenicity island termed locus of enterocyte effacement (LEE) responsible for attaching and effacing lesions on epithelial cells. The expression of LEE varies among isolates and is dependent on environmental cues. In the EHEC O157:H7 Sakaï isolate (RIMD-0509952 strain), we found that the non-coding RNA, DsrA, activates the expression of the LEE. This activation requires RpoS, the stress sigma factor. The DsrA/RpoS regulatory pathway mediates its positive effect by stimulating the transcription of ler, a positive regulatory gene encoded by the LEE. A second regulatory pathway, repressed by HNS, is also able to activate the transcription of ler and requires GrlA, another LEE-encoded regulator. Both regulatory pathways, DsrA/RpoS and HNS/GrlA, affect the activity of the ler distal promoter and require the Ler protein to be functional. Our data demonstrate that the LEE expression can be turned on by at least two separate pathways acting on the transcription of ler.

Construction and evaluation of a chromosomal expression platform (CEP) for ectopic, maltose-driven gene expression in Streptococcus pneumoniae.

In this paper, the construction and evaluation of a chromosomal expression platform (CEP), which allows controlled gene expression following ectopic integration into the chromosome of Streptococcus pneumoniae, is described. CEP is based on the well-studied maltosaccharide-inducible system. To facilitate integration at CEP, a plasmid, pCEP, capable of replication in Escherichia coli, but not in S. pneumoniae, was assembled. This plasmid contains an expression/selection cassette flanked on each side by more than 2 kb of pneumococcal DNA. The cassette comprises a maltose-inducible promoter, P(M), separated from a kanamycin-resistance gene by NcoI and BamHI cloning sites. Clones harbouring the gene of interest integrated at CEP under the control of P(M) can be obtained through direct transformation of an S. pneumoniae recipient with ligation products between that gene and NcoI/BamHI-digested pCEP DNA, followed by selection for kanamycin-resistant transformants.