Emil Lesho

A novel method of consensus pan-chromosome assembly and large-scale comparative analysis reveal the highly flexible pan-genome of Acinetobacter baumannii

BackgroundInfections by pan-drug resistant Acinetobacter baumannii plague military and civilian healthcare systems. Previous A. baumannii pan-genomic studies used modest sample sizes of low diversity and comparisons to a single reference genome, limiting our understanding of gene order and content. A consensus representation of multiple genomes will provide a better framework for comparison. A large-scale comparative study will identify genomic determinants associated with their diversity and adaptation as a successful pathogen.ResultsWe determine draft-level genomic sequence of 50 diverse military isolates and conduct the largest bacterial pan-genome analysis of 249 genomes. The pan-genome of A. baumannii is open when the input genomes are normalized for diversity with 1867 core proteins and a paralog-collapsed pan-genome size of 11,694 proteins. We developed a novel graph-based algorithm and use it to assemble the first consensus pan-chromosome, identifying both the order and orientation of core genes and flexible genomic regions. Comparative genome analyses demonstrate the existence of novel resistance islands and isolates with increased numbers of resistance island insertions over time, from single insertions in the 1950s to triple insertions in 2011. Gene clusters responsible for carbon utilization, siderophore production, and pilus assembly demonstrate frequent gain or loss among isolates.ConclusionsThe highly variable and dynamic nature of the A. baumannii genome may be the result of its success in rapidly adapting to both abiotic and biotic environments through the gain and loss of gene clusters controlling fitness. Importantly, some archaic adaptation mechanisms appear to have reemerged among recent isolates.

Escherichia coli Harboring mcr-1 and blaCTX-M on a Novel IncF Plasmid: First Report of mcr-1 in the United States

The recent discovery of a plasmid-borne colistin resistance gene, mcr-1 , in China heralds the emergence of truly pan-drug-resistant bacteria ([1][1]). The gene has been found primarily in Escherichia coli but has also been identified in other members of the Enterobacteriaceae in human, animal, food

Emergence of Colistin-Resistance in Extremely Drug-Resistant Acinetobacter baumannii Containing a Novel pmrCAB Operon During Colistin Therapy of Wound Infections

BACKGROUND Colistin resistance is of concern since it is increasingly needed to treat infections caused by bacteria resistant to all other antibiotics and has been associated with poorer outcomes. Longitudinal data from in vivo series are sparse. METHODS Under a quality-improvement directive to intensify infection-control measures, extremely drug-resistant (XDR) bacteria undergo phenotypic and molecular analysis. RESULTS Twenty-eight XDR Acinetobacter baumannii isolates were longitudinally recovered during colistin therapy. Fourteen were susceptible to colistin, and 14 were resistant to colistin. Acquisition of colistin resistance did not alter resistance to other antibiotics. Isolates had low minimum inhibitory concentrations of an investigational aminoglycoside, belonged to multi-locus sequence type 94, were indistinguishable by pulsed-field gel electrophoresis and optical mapping, and harbored a novel pmrC1A1B allele. Colistin resistance was associated with point mutations in the pmrA1 and/or pmrB genes. Additional pmrC homologs, designated eptA-1 and eptA-2, were at distant locations from the operon. Compared with colistin-susceptible isolates, colistin-resistant isolates displayed significantly enhanced expression of pmrC1A1B, eptA-1, and eptA-2; lower growth rates; and lowered fitness. Phylogenetic analysis suggested that colistin resistance emerged from a single progenitor colistin-susceptible isolate. CONCLUSIONS We provide insights into the in vivo evolution of colistin resistance in a series of XDR A. baumannii isolates recovered during therapy of infections and emphasize the importance of antibiotic stewardship and surveillance.

Detection of Bacterial 16S rRNA and Identification of Four Clinically Important Bacteria by Real-Time PCR

Within the paradigm of clinical infectious disease research, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa represent the four most clinically relevant, and hence most extensively studied bacteria. Current culture-based methods for identifying these organisms are slow and cumbersome, and there is increasing need for more rapid and accurate molecular detection methods. Using bioinformatic tools, 962,279 bacterial 16S rRNA gene sequences were aligned, and regions of homology were selected to generate a set of real-time PCR primers that target 93.6% of all bacterial 16S rRNA sequences published to date. A set of four species-specific real-time PCR primer pairs were also designed, capable of detecting less than 100 genome copies of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa. All primers were tested for specificity in vitro against 50 species of Gram-positive and –negative bacteria. Additionally, the species-specific primers were tested against a panel of 200 clinical isolates of each species, randomly selected from a large repository of clinical isolates from diverse areas and sources. A comparison of culture and real-time PCR demonstrated 100% concordance. The primers were incorporated into a rapid assay capable of positive identification from plate or broth cultures in less than 90 minutes. Furthermore, our data demonstrate that current targets, such as the uidA gene in E.coli, are not suitable as species-specific genes due to sequence variation. The assay described herein is rapid, cost-effective and accurate, and can be easily incorporated into any research laboratory capable of real-time PCR.

Complete sequence of a novel 178-kilobase plasmid carrying bla(NDM-1) in a Providencia stuartii strain isolated in Afghanistan.

ABSTRACT In response to global concerns over the spread of the New Delhi metallo-β-lactamase gene 1, blaNDM-1, a monthly surveillance program was initiated in September 2010. All carbapenem-resistant Gram-negative strains forwarded to our facility are screened for this gene. To date, 321 carbapenem-resistant isolates, encompassing 11 bacterial species, have been tested. In February 2011, two strains of Providencia stuartii, submitted from a military hospital in Afghanistan, tested positive for blaNDM-1. Both strains were identical by pulsed-field gel electrophoresis (PFGE). blaNDM-1 was carried on a large plasmid, pMR0211, which was sequenced by emulsion PCR and pyrosequencing. pMR0211 is 178,277 bp in size and belongs to incompatibility group A/C. The plasmid consists of a backbone with considerable homology to pAR060302 from Escherichia coli, and it retains many of the antibiotic resistance genes associated with it. The plasmid also shares common elements with the pNDM-HK plasmid, including blaNDM-1, armA, and sul1. However, gene orientation is reversed, and a 3-kb fragment from this region is absent from pMR0211. pMR0211 also contains additional genes, including the aminoglycoside-modifying enzyme loci aadA and aac(6′), the quinolone resistance gene qnrA, a gene with highest homology to a U32 family peptidase from Shewanella amazonensis, and the blaOXA-10 gene. The finding of this gene in an intrinsically colistin-resistant species such as Providencia stuartii is especially worrisome, as it renders the organism resistant to nearly every available antibiotic. The presence of multiple insertion sequences and transposons flanking the region containing the blaNDM-1 gene further highlights the potential mobility associated with this gene.

AB5075, a Highly Virulent Isolate of Acinetobacter baumannii, as a Model Strain for the Evaluation of Pathogenesis and Antimicrobial Treatments

ABSTRACT Acinetobacter baumannii is recognized as an emerging bacterial pathogen because of traits such as prolonged survival in a desiccated state, effective nosocomial transmission, and an inherent ability to acquire antibiotic resistance genes. A pressing need in the field of A. baumannii research is a suitable model strain that is representative of current clinical isolates, is highly virulent in established animal models, and can be genetically manipulated. To identify a suitable strain, a genetically diverse set of recent U.S. military clinical isolates was assessed. Pulsed-field gel electrophoresis and multiplex PCR determined the genetic diversity of 33 A. baumannii isolates. Subsequently, five representative isolates were tested in murine pulmonary and Galleria mellonella models of infection. Infections with one strain, AB5075, were considerably more severe in both animal models than those with other isolates, as there was a significant decrease in survival rates. AB5075 also caused osteomyelitis in a rat open fracture model, while another isolate did not. Additionally, a Tn5 transposon library was successfully generated in AB5075, and the insertion of exogenous genes into the AB5075 chromosome via Tn7 was completed, suggesting that this isolate may be genetically amenable for research purposes. Finally, proof-of-concept experiments with the antibiotic rifampin showed that this strain can be used in animal models to assess therapies under numerous parameters, including survival rates and lung bacterial burden. We propose that AB5075 can serve as a model strain for A. baumannii pathogenesis due to its relatively recent isolation, multidrug resistance, reproducible virulence in animal models, and genetic tractability. IMPORTANCE The incidence of A. baumannii infections has increased over the last decade, and unfortunately, so has antibiotic resistance in this bacterial species. A. baumannii is now responsible for more than 10% of all hospital-acquired infections in the United States and has a >50% mortality rate in patients with sepsis and pneumonia. Most research on the pathogenicity of A. baumannii focused on isolates that are not truly representative of current multidrug-resistant strains isolated from patients. After screening of a panel of isolates in different in vitro and in vivo assays, the strain AB5075 was selected as more suitable for research because of its antibiotic resistance profile and increased virulence in animal models. Moreover, AB5075 is susceptible to tetracycline and hygromycin, which makes it amenable to genetic manipulation. Taken together, these traits make AB5075 a good candidate for use in studying virulence and pathogenicity of this species and testing novel antimicrobials. The incidence of A. baumannii infections has increased over the last decade, and unfortunately, so has antibiotic resistance in this bacterial species. A. baumannii is now responsible for more than 10% of all hospital-acquired infections in the United States and has a >50% mortality rate in patients with sepsis and pneumonia. Most research on the pathogenicity of A. baumannii focused on isolates that are not truly representative of current multidrug-resistant strains isolated from patients. After screening of a panel of isolates in different in vitro and in vivo assays, the strain AB5075 was selected as more suitable for research because of its antibiotic resistance profile and increased virulence in animal models. Moreover, AB5075 is susceptible to tetracycline and hygromycin, which makes it amenable to genetic manipulation. Taken together, these traits make AB5075 a good candidate for use in studying virulence and pathogenicity of this species and testing novel antimicrobials.

Clinical Use of Colistin Induces Cross-Resistance to Host Antimicrobials in Acinetobacter baumannii

ABSTRACT The alarming rise in antibiotic resistance has led to an increase in patient mortality and health care costs. This problem is compounded by the absence of new antibiotics close to regulatory approval. Acinetobacter baumannii is a human pathogen that causes infections primarily in patients in intensive care units (ICUs) and is highly antibiotic resistant. Colistin is one of the last-line antibiotics for treating A. baumannii infections; however, colistin-resistant strains are becoming increasingly common. This cationic antibiotic attacks negatively charged bacterial membranes in a manner similar to that seen with cationic antimicrobials of the innate immune system. We therefore set out to determine if the increasing use of colistin, and emergence of colistin-resistant strains, is concomitant with the generation of cross-resistance to host cationic antimicrobials. We found that there is indeed a positive correlation between resistance to colistin and resistance to the host antimicrobials LL-37 and lysozyme among clinical isolates. Importantly, isolates obtained before and after treatment of individual patients demonstrated that colistin use correlated with increased resistance to cationic host antimicrobials. These data reveal the overlooked risk of inducing cross-resistance to host antimicrobials when treating patients with colistin as a last-line antibiotic. IMPORTANCE Increased use of the cationic antibiotic colistin to treat multidrug-resistant Acinetobacter baumannii has led to the development of colistin-resistant strains. Here we report that treatment of patients with colistin can induce not only increased resistance to colistin but also resistance to host cationic antimicrobials. This worrisome finding likely represents an example of a broader trend observed in other bacteria against which colistin is used therapeutically such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Furthermore, these data suggest that the possible future use of an array of cationic antimicrobial peptides in development as therapeutics may have unintended negative consequences, eventually leading to the generation of hypervirulent strains that are resistant to innate host defenses. The potential for the induction of cross-resistance to innate immune antimicrobials should be considered during the development of new therapeutics. Increased use of the cationic antibiotic colistin to treat multidrug-resistant Acinetobacter baumannii has led to the development of colistin-resistant strains. Here we report that treatment of patients with colistin can induce not only increased resistance to colistin but also resistance to host cationic antimicrobials. This worrisome finding likely represents an example of a broader trend observed in other bacteria against which colistin is used therapeutically such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Furthermore, these data suggest that the possible future use of an array of cationic antimicrobial peptides in development as therapeutics may have unintended negative consequences, eventually leading to the generation of hypervirulent strains that are resistant to innate host defenses. The potential for the induction of cross-resistance to innate immune antimicrobials should be considered during the development of new therapeutics.

Evidence base for using corticosteroids to treat HIV-associated immune reconstitution syndrome.

Most of the evidence supporting the use of corticosteroids (steroids) for immune reconstitution syndrome (IRS) comes from case reports or retrospective series and is of low quality. However, when steroids are used, they have usually been associated with clinical improvement or resolution of IRS. Except in the case of hepatitis B- or C-associated IRS, there have been no reports of worsening of the IRS or adverse outcome due to steroid use. After ruling out other conditions which can mimic IRS, clinicians should strongly consider steroids when managing IRS associated with mycobacterial or fungal pathogens when there is severe disease, or when other measures have failed.

Amplification of Aminoglycoside Resistance Gene aphA1 in Acinetobacter baumannii Results in Tobramycin Therapy Failure

ABSTRACT Gene amplification is believed to play an important role in antibiotic resistance but has been rarely documented in clinical settings because of its unstable nature. We report a rise in MICs from 0.5 to 16 μg/ml in successive Acinetobacter baumannii isolated over 4 days from a patient being treated with tobramycin for an infection by multidrug-resistant A. baumannii, resulting in therapeutic failure. Isolates were characterized by whole-genome sequencing, real-time and reverse transcriptase PCR, and growth assays to determine the mechanism of tobramycin resistance and its fitness cost. Tobramycin resistance was associated with two amplification events of different chromosomal fragments containing the aphA1 aminoglycoside resistance gene part of transposon Tn6020. The first amplification event involved low amplification (6 to 10 copies) of a large DNA fragment that was unstable and conferred tobramycin MICs of ≤8 μg/ml. The second event involved moderate (10 to 30 copies) or high (40 to 110 copies) amplification of Tn6020. High copy numbers were associated with tobramycin MICs of 16 μg/ml, impaired fitness, and genetic instability, whereas lower copy numbers resulted in tobramycin MICs of ≤8 μg/ml and no fitness cost and were stably maintained in vitro. Exposure in vitro to tobramycin of the initial susceptible isolate and of the A. baumannii AB0057 reference strain led to similar aphA1 amplifications and elevated tobramycin MICs. To the best of our knowledge, this is the first report of in vivo development of antibiotic resistance secondary to gene amplifications resulting in therapy failure. IMPORTANCE A combination of whole-genome sequencing and mapping were used to detect an antibiotic resistance mechanism, gene amplification, which has been presumed for a long time to be of major importance but has rarely been reported in clinical settings because of its unstable nature. Two gene amplification events in a patient with an Acinetobacter baumannii infection treated with tobramycin were identified. One gene amplification event led to high levels of resistance and was rapidly reversible, while the second event led to low and more stable resistance since it incurred low fitness cost on the host. Gene amplification, with an associated rise in tobramycin MICs, could be readily reproduced in vitro from initially susceptible strains exposed to increasing concentrations of tobramycin, suggesting that gene amplification in A. baumannii may be a more common mechanism than currently believed. This report underscores the importance of rapid molecular techniques for surveillance of drug resistance. A combination of whole-genome sequencing and mapping were used to detect an antibiotic resistance mechanism, gene amplification, which has been presumed for a long time to be of major importance but has rarely been reported in clinical settings because of its unstable nature. Two gene amplification events in a patient with an Acinetobacter baumannii infection treated with tobramycin were identified. One gene amplification event led to high levels of resistance and was rapidly reversible, while the second event led to low and more stable resistance since it incurred low fitness cost on the host. Gene amplification, with an associated rise in tobramycin MICs, could be readily reproduced in vitro from initially susceptible strains exposed to increasing concentrations of tobramycin, suggesting that gene amplification in A. baumannii may be a more common mechanism than currently believed. This report underscores the importance of rapid molecular techniques for surveillance of drug resistance.

Novel 16S rRNA Methyltransferase RmtH Produced by Klebsiella pneumoniae Associated with War-Related Trauma

ABSTRACT Klebsiella pneumoniae strain MRSN2404 was isolated from the chronic wound of a soldier who had been wounded in Iraq in 2006. The strain displayed very high MICs of all aminoglycosides, including arbekacin. A gene encoding a novel 16S rRNA methyltransferase, now designated RmtH, was identified. RmtH had 64% identity with RmtB1 and RmtB2. rmtH was bracketed by two copies of ISCR2, which may have played a role in its mobilization.