Dataset Information

Fluorescence-based detection of natural transformation in drug resistant Acinetobacter baumannii.

ABSTRACT: 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 method. 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 non-clinical 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 pan-resistant 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 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.

SUBMITTER: Godeux AS

PROVIDER: S-EPMC6148472 | biostudies-other | 2018 Jul

REPOSITORIES: biostudies-other

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Publications

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

Godeux Anne-Sophie AS Lupo Agnese A Haenni Marisa M Guette-Marquet Simon S Wilharm Gottfried G Laaberki Maria-Halima MH Charpentier Xavier X

Journal of bacteriology 20180910 19

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 ...[more]

PMID: 30012729

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Project description:Carbapenem-resistant Acinetobacter baumannii (CRAB) is an important opportunistic pathogen linked to a variety of nosocomial infections and hospital outbreaks worldwide. This study aimed at investigating and characterizing a CRAB outbreak at a large tertiary hospital in Lebanon. A total of 41 isolates were collected and analyzed using pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing (WGS) was performed on all the isolates, and long-read PacBio sequencing was used to generate reference genomes. The multilocus sequence types (MLST), repertoire of resistance genes, and virulence factors were determined from the sequencing data. The plasmid content was analyzed both in silico and using the A. baumannii PCR-based replicon typing (AB-PBRT) method. Genome analysis initially revealed two clones, one carrying bla OXA-23 on Tn2006 (ST-1305, ST-195, and ST-218) and another carrying bla OXA-72 on pMAL-1 (ST-502 and ST-2059, a new ST), with the latter having two subclones, as revealed using the Bayesian transmission network. All isolates were extensively drug resistant (XDR). WGS analysis revealed the transmission pathways and demonstrated the diversity of CRAB isolates and mobile genetic elements in this health care setting. Outbreak detection using WGS and immediate implementation of infection control measures contribute to restraining the spread and decreasing mortality.IMPORTANCE Carbapenem-resistant Acinetobacter baumannii (CRAB) has been implicated in hospital outbreaks worldwide. Here, we present a whole-genome-based investigation of an extensively drug-resistant CRAB outbreak rapidly spreading and causing high incidences of mortality at numerous wards of a large tertiary hospital in Lebanon. This is the first study of its kind in the region. Two circulating clones were identified using a combination of molecular typing approaches, short- and long-read sequencing and Bayesian transmission network analysis. One clone carried bla OXA-23 on Tn2006 (ST-1305, ST-195, and ST-218), and another carried bla OXA-72 on a pMAL-1 plasmid (ST-502 and ST-2059, a new ST). A pMAL-2 plasmid was circulating between the two clones. The approaches implemented in this study and the obtained findings facilitate the tracking of outbreak scenarios in Lebanon and the region at large.

Project description:Acinetobacter baumannii is a severe threat to human health as a frequently multidrug-resistant hospital-acquired pathogen. Part of the danger from this bacterium comes from its genome plasticity and ability to evolve quickly by taking up and recombining external DNA into its own genome in a process called natural competence for transformation. This mode of horizontal gene transfer is one of the major ways that bacteria can acquire new antimicrobial resistances and toxic traits. Because these processes in A. baumannii are not well studied, we herein characterized new aspects of natural transformability in this species that include the species' competence window. We uncovered a strong correlation with a growth phase-dependent synthesis of a type IV pilus (TFP), which constitutes the central part of competence-induced DNA uptake machinery. We used bacterial genetics and microscopy to demonstrate that the TFP is essential for the natural transformability and surface motility of A. baumannii, whereas pilus-unrelated proteins of the DNA uptake complex do not affect the motility phenotype. Furthermore, TFP biogenesis and assembly is subject to input from two regulatory systems that are homologous to Pseudomonas aeruginosa, namely, the PilSR two-component system and the Pil-Chp chemosensory system. We demonstrated that these systems affect not only the piliation status of cells but also their ability to take up DNA for transformation. Importantly, we report on discrepancies between TFP biogenesis and natural transformability within the same genus by comparing data for our work on A. baumannii to data reported for Acinetobacter baylyi, the latter of which served for decades as a model for natural competence.IMPORTANCE Rapid bacterial evolution has alarming negative impacts on animal and human health which can occur when pathogens acquire antimicrobial resistance traits. As a major cause of antibiotic-resistant opportunistic infections, A. baumannii is a high-priority health threat which has motivated renewed interest in studying how this pathogen acquires new, dangerous traits. In this study, we deciphered a specific time window in which these bacteria can acquire new DNA and correlated that with its ability to produce the external appendages that contribute to the DNA acquisition process. These cell appendages function doubly for motility on surfaces and for DNA uptake. Collectively, we showed that A. baumannii is similar in its TFP production to Pseudomonas aeruginosa, though it differs from the well-studied species A. baylyi.

Project description:BackgroundDetection of Acinetobacter baumannii has been relying primarily on bacterial culture that often fails to return useful results in time. Although DNA-based assays are more sensitive than bacterial culture in detecting the pathogen, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. In addition, these molecular tools require expensive laboratory instruments. Therefore, establishing molecular tools for field use require simpler molecular platforms. The loop-mediated isothermal amplification method is relatively simple and can be improved for better use in a routine clinical bacteriology laboratory. A simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in the same platform has been developed in recent years. This method is referred to as real-time loop-mediated isothermal amplification. In this study, we attempted to utilize this method for rapid detection of A. baumannii.Methodology and significant findingsSpecies-specific primers were designed to test the utility of this method. Clinical samples of A. baumannii were used to determine the sensitivity and specificity of this system compared to bacterial culture and a polymerase chain reaction method. All positive samples isolated from sputum were confirmed to be the species of Acinetobacter by 16S rRNA gene sequencing. The RealAmp method was found to be simpler and allowed real-time detection of DNA amplification, and could distinguish A. baumannii from Acinetobacter calcoaceticus and Acinetobacter genomic species 3. DNA was extracted by simple boiling method. Compared to bacterial culture, the sensitivity and specificity of RealAmp in detecting A. baumannii was 98.9% and 75.0%, respectively.ConclusionThe RealAmp assay only requires a single unit, and the assay positivity can be verified by visual inspection. Therefore, this assay has great potential of field use as a molecular tool for detection of A. baumannii.

Project description:Nosocomial infections with Acinetobacter baumannii are a global problem in intensive care units with high mortality rates. Increasing resistance to first- and second-line antibiotics has forced the use of colistin as last-resort treatment, and increasing development of colistin resistance in A. baumannii has been reported. We evaluated the transcriptional regulator PmrA as potential drug target to restore colistin efficacy in A. baumannii Deletion of pmrA restored colistin susceptibility in 10 of the 12 extensively drug-resistant A. baumannii clinical isolates studied, indicating the importance of PmrA in the drug resistance phenotype. However, two strains remained highly resistant, indicating that PmrA-mediated overexpression of the phosphoethanolamine (PetN) transferase PmrC is not the exclusive colistin resistance mechanism in A. baumannii A detailed genetic characterization revealed a new colistin resistance mechanism mediated by genetic integration of the insertion element ISAbaI upstream of the PmrC homolog EptA (93% identity), leading to its overexpression. We found that eptA was ubiquitously present in clinical strains belonging to the international clone 2, and ISAbaI integration upstream of eptA was required to mediate the colistin-resistant phenotype. In addition, we found a duplicated ISAbaI-eptA cassette in one isolate, indicating that this colistin resistance determinant may be embedded in a mobile genetic element. Our data disprove PmrA as a drug target for adjuvant therapy but highlight the importance of PetN transferase-mediated colistin resistance in clinical strains. We suggest that direct targeting of the homologous PetN transferases PmrC/EptA may have the potential to overcome colistin resistance in A. baumanniiIMPORTANCE The discovery of antibiotics revolutionized modern medicine and enabled us to cure previously deadly bacterial infections. However, a progressive increase in antibiotic resistance rates is a major and global threat for our health care system. Colistin represents one of our last-resort antibiotics that is still active against most Gram-negative bacterial pathogens, but increasing resistance is reported worldwide, in particular due to the plasmid-encoded protein MCR-1 present in pathogens such as Escherichia coli and Klebsiella pneumoniae Here, we showed that colistin resistance in A. baumannii, a top-priority pathogen causing deadly nosocomial infections, is mediated through different avenues that result in increased activity of homologous phosphoethanolamine (PetN) transferases. Considering that MCR-1 is also a PetN transferase, our findings indicate that PetN transferases might be the Achilles heel of superbugs and that direct targeting of them may have the potential to preserve the activity of polymyxin antibiotics.

Dataset Information

Fluorescence-based detection of natural transformation in drug resistant Acinetobacter baumannii.

Publications

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

Similar Datasets

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