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Defining gene-phenotype relationships in Acinetobacter baumannii through one-step chromosomal gene inactivation.

ABSTRACT: Rates of infection with hospital-acquired Acinetobacter baumannii have exploded over the past decade due to our inability to limit persistence and effectively treat disease. A. baumannii quickly acquires antibiotic resistance, and its genome encodes mechanisms to tolerate biocides and desiccation, which enhance its persistence in hospital settings. With depleted antibiotic options, new methods to treat A. baumannii infections are desperately needed. A comprehensive understanding detailing A. baumannii cellular factors that contribute to its resiliency at genetic and mechanistic levels is vital to the development of new treatment options. Tools to rapidly dissect the A. baumannii genome will facilitate this goal by quickly advancing our understanding of A. baumannii gene-phenotype relationships. We describe here a recombination-mediated genetic engineering (recombineering) system for targeted genome editing of A. baumannii. We have demonstrated that this system can perform directed mutagenesis on wide-ranging genes and operons and is functional in various strains of A. baumannii, indicating its broad application. We utilized this system to investigate key gene-phenotype relationships in A. baumannii biology important to infection and persistence in hospitals, including oxidative stress protection, biocide resistance mechanisms, and biofilm formation. In addition, we have demonstrated that both the formation and movement of type IV pili play an important role in A. baumannii biofilm. Importance: Acinetobacter baumannii is the causative agent of hospital-acquired infections, including pneumonia and serious blood and wound infections. A. baumannii is an emerging pathogen and has become a threat to public health because it quickly develops antibiotic resistance, making treatment difficult or impossible. While the threat of A. baumannii is well recognized, our understanding of even its most basic biology lags behind. Analysis of A. baumannii cellular functions to identify potential targets for drug development has stalled due in part to laborious genetic techniques. Here we have pioneered a novel recombineering system that facilitates efficient genome editing in A. baumannii by single PCR products. This technology allows for rapid genome editing to quickly ascertain gene-phenotype relationships. To demonstrate the power of recombineering in dissecting A. baumannii biology, we use this system to establish key gene-phenotype relationships important to infection and persistence in hospitals, including oxidative stress protection, biocide resistance, and biofilm formation.

SUBMITTER: Tucker AT

PROVIDER: S-EPMC4128354 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Defining gene-phenotype relationships in Acinetobacter baumannii through one-step chromosomal gene inactivation.

Tucker Ashley T AT Nowicki Emily M EM Boll Joseph M JM Knauf Gregory A GA Burdis Nora C NC Trent M Stephen MS Davies Bryan W BW

mBio 20140805 4

Rates of infection with hospital-acquired Acinetobacter baumannii have exploded over the past decade due to our inability to limit persistence and effectively treat disease. A. baumannii quickly acquires antibiotic resistance, and its genome encodes mechanisms to tolerate biocides and desiccation, which enhance its persistence in hospital settings. With depleted antibiotic options, new methods to treat A. baumannii infections are desperately needed. A comprehensive understanding detailing A. bau ...[more]

PMID: 25096877

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Project description:The opportunistic pathogen A. baumannii has a remarkable capacity to persist in the hospital environment and cause devastating human infections. This capacity can be attributed partly to the sensing and regulatory systems that enable this pathogen to modify its physiology based on environmental cues. One of the signals that A. baumannii senses and responds to is light through the sensing and regulatory roles of the BlsA photoreceptor protein in cells cultured at temperatures below 30°C. This report presents evidence that a light stimulon is operational at 37°C, a condition at which the BlsA production and activity are drastically impaired. Global transcriptional analysis showed that the 37°C light stimulon includes the differential expression of chromosomal genes encoding a wide range of functions that are known to be involved in the adaptation to different metabolic conditions, as well as virulence and persistence in the host and the medical environment. Unexpectedly, the 37°C light stimulon also includes the differential expression of conjugation functions encoded by pAB3 plasmid genes. Our work further demonstrates that the TetR1 and H-NS regulators encoded by this conjugative plasmid control the expression of H2O2 resistance and surface motility, respectively. Furthermore, our data showed that pAB3 has an overall negative effect on the expression of these phenotypes and plays no significant virulence role. Although the nature of the bacterial factors and the mechanisms by which the regulation is attained at 37°C remain unknown, taken together, our work expands the current knowledge about light sensing and gene regulation in A. baumannii. IMPORTANCE As a facultative pathogen, Acinetobacter baumannii persists in various environments by sensing different environmental cues, including light. This report provides evidence of light-dependent regulation at 37°C of the expression of genes coding for a wide range of functions, including those involved in the conjugation of the pAB3 plasmid. Although this plasmid affects the expression of virulence traits when tested under laboratory conditions, it does not have a significant impact when tested using ex vivo and in vivo experimental models. These findings provide a better understanding of the interplay between light regulation and plasmid persistence in the pathobiology of A. baumannii.

Project description:Abstract Background Efficacy of Colistin the last line agent against infections due to multidrug-resistant (MDR) gram-negative pathogens, has been challenged when Liu et al. reported a plasmid-mediated gene, mcr-1, in 2015. Thereby this plasmid-borne mcr has been reported in bacterial isolates worldwide taken from humans, animals, farms, foods, and the environment. The present work invesitigate the mcr gene among clinical isolates of Acinetobacter Baumannii at our tertiary referral hospital of India. Methods The study was conducted at Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India. MIC values for 100 consecutive non-duplicate MDR isolates of Acinetobacter were checked for Colistin. PCR amplification of mcr gene was performed followed by sequencing of the amplicons. Clinical features of patients infected with mcr positive isolates were unveiled. Clonal relatedness of these isolates was investigated by Pulsed-field gel electrophoresis (PFGE). The mcr-1 localization was checked by conjugation followed by PFGE southern hybridization. Results 20/100 (20%) isolates were colistin resistant with having MIC Values of more than 8? µg/mL. The 20 colistin resistances isolates were PCR positive for mcr-1 and had been assigned EMBL/GeneBank nucleotide accession numbers MH730099-MH730118. Oher antibiotic resistance gene like ESBL, NDM-1, VIM, and 16s rRNA methyl transferases like Arm A, rmtC, rmt F were also found in these isolates. Majority of these patients recovered from the infection (65%) after proper antibiotic therapy.The ISApl1 transposable elements were not detected in these isolates. These isolates were found clonally unrelated when analyze by pulsed-field gel electrophoresis. The conjugation attempt to transfer mcr-1 to recipient’s E. coli J53 failed, Southern hybridization showed that mcr-1 was found located on chromosome in multiple copies. Conclusion This is the first case of mcr-1 in a human clinical isolate in Acinetobacter Baumnnii from India. These findings highlight the vertical transferability of colistin resistance by mcr-1 gene in Acinetobacter Baumnnii with the association of known some unknown insertion sequence located on chromosome. Strategies required to contain their spread and evolution of such genes. Disclosures All authors: No reported disclosures.

Dataset Information

Defining gene-phenotype relationships in Acinetobacter baumannii through one-step chromosomal gene inactivation.

Publications

Defining gene-phenotype relationships in Acinetobacter baumannii through one-step chromosomal gene inactivation.

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