Yoon I. Kwak

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.

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.

Detection of qacA/B in Clinical Isolates of Methicillin-Resistant Staphylococcus aureus from a Regional Healthcare Network in the Eastern United States

We describe the clinical, microbiologic, and molecular features of the first series of qacA/B-containing strains of methicillin-resistant Staphylococcus aureus from infected US patients. All qac-carrying strains were clonally diverse, and qacA strains exhibited increased tolerance to chlorhexidine as measured by minimum inhibitory concentrations, minimum bactericidal concentrations, and postexposure colony counts.

Fatal Outbreak of an Emerging Clone of Extensively Drug-Resistant Acinetobacter baumannii With Enhanced Virulence

BACKGROUND Severe Acinetobacter baumannii infections in immunocompetent patients are uncommon, and the virulence mechanisms of this organism are not fully understood. METHODS Following an outbreak of fatal A. baumannii infections in a cohort of relatively immunocompetent patients (low comorbidity and illness severity scores), isolates were investigated with comparative genomics and in animal models. RESULTS Two unrelated A. baumannii clades were associated with the outbreak. The clone associated with the majority of patient deaths, clade B, is evolutionarily distinct from the 3 international clonal complexes, belongs to multilocus sequence type (MLST) 10, and is most closely related to strains isolated from the Czech Republic, California, and Germany in 1994, 1997, and 2003, respectively. In 2 different murine models, clade B isolates were more virulent than comparator strains, including the highly virulent reference strain AB5075. The most virulent clade B derivative, MRSN 16897, was isolated from the patient with the lowest combined comorbidity/illness severity score. Clade B isolates possess a unique combination of putative virulence genes involved in iron metabolism, protein secretion, and glycosylation, which was leveraged to develop a rapid and specific clinical assay to detect this clade that cannot be distinguished by MLST. CONCLUSIONS Clade B warrants continued surveillance and investigation.

Rapid and Simultaneous Detection of blaKPC and blaNDM by Use of Multiplex Real-Time PCR

ABSTRACT The increasing incidence of carbapenem nonsusceptibility among clinically important species is of global concern. Identification of the molecular mechanisms underlying carbapenem nonsusceptibility is critical for epidemiological investigations. In this report, we describe a real-time PCR-based assay capable of simultaneously detecting bla KPC and bla NDM, two of the most important carbapenemases, directly from culture in less than 90 min. The assay was validated with bla KPC- and bla NDM-carrying clinical isolates and demonstrated 100% concordance with the Carba NP test.

IS5 Element Integration, a Novel Mechanism for Rapid In Vivo Emergence of Tigecycline Nonsusceptibility in Klebsiella pneumoniae

ABSTRACT Tigecycline nonsusceptibility is concerning because tigecycline is increasingly relied upon to treat carbapenem- or colistin-resistant organisms. In Enterobacteriaceae, tigecycline nonsusceptibility is mediated by the AcrAB-TolC efflux pump, among others, and pump activity is often a downstream effect of mutations in their transcriptional regulators, cognate repressor genes, or noncoding regions, as demonstrated in Enterobacteriaceae and Acinetobacter isolates. Here, we report the emergence of tigecycline nonsusceptibility in a longitudinal series of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Klebsiella pneumoniae isolates collected during tigecycline therapy and the elucidation of its resistance mechanisms. Clinical isolates were recovered prior to and during tigecycline therapy of a 2.5-month-old Honduran neonate. Antimicrobial susceptibility tests to tigecycline determined that the MIC increased from 1 to 4 μg/ml prior to the completion of tigecycline therapy. Unlike other studies, we did not find increased expression of ramA, ramR, oqxA, acrB, marA, or rarA genes by reverse transcription-quantitative PCR (qRT-PCR). Whole-genome sequencing revealed an IS5 insertion element in nonsusceptible isolates 85 bp upstream of a putative efflux pump operon, here named kpgABC, previously unknown to be involved in resistance. Introduction of the kpgABC genes in a non-kpgABC background increased the MIC of tigecycline 4-fold and is independent of a functional AcrAB-TolC pump. This is the first report to propose a function for kpgABC and identify an insertion element whose presence correlated with the in vivo development of tigecycline nonsusceptibility in K. pneumoniae.

Rapid and Simultaneous Detection of blaKPC and blaNDM using Multiplex Real-Time PCR

ABSTRACT The increasing incidence of carbapenem nonsusceptibility among clinically important species is of global concern. Identification of the molecular mechanisms underlying carbapenem nonsusceptibility is critical for epidemiological investigations. In this report, we describe a real-time PCR-based assay capable of simultaneously detecting bla KPC and bla NDM, two of the most important carbapenemases, directly from culture in less than 90 min. The assay was validated with bla KPC- and bla NDM-carrying clinical isolates and demonstrated 100% concordance with the Carba NP test.