Robert Clifford

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.

Comparison of Global Gene Expression of Gastric Cardia and Noncardia Cancers from a High-Risk Population in China

Objective To profile RNA expression in gastric cancer by anatomic subsites as an initial step in identifying molecular subtypes and providing targets for early detection and therapy. Methods We performed transcriptome analysis using the Affymetrix GeneChip U133A in gastric cardia adenocarcinomas (n = 62) and gastric noncardia adenocarcinomas (n = 72) and their matched normal tissues from patients in Shanxi Province, and validated selected dysregulated genes with additional RNA studies. Expression of dysregulated genes was also related to survival of cases. Results Principal Component Analysis showed that samples clustered by tumor vs. normal, anatomic location, and histopathologic features. Paired t-tests of tumor/normal tissues identified 511 genes whose expression was dysregulated (P<4.7E-07 and at least two-fold difference in magnitude) in cardia or noncardia gastric cancers, including nearly one-half (n = 239, 47%) dysregulated in both cardia and noncardia, one-fourth dysregulated in cardia only (n = 128, 25%), and about one-fourth in noncardia only (n = 144, 28%). Additional RNA studies confirmed profiling results. Expression was associated with case survival for 20 genes in cardia and 36 genes in noncardia gastric cancers. Conclusions The dysregulated genes identified here represent a comprehensive starting point for future efforts to understand etiologic heterogeneity, develop diagnostic biomarkers for early detection, and test molecularly-targeted therapies for gastric cancer.

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.

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.

Enhanced de novo assembly of high throughput pyrosequencing data using whole genome mapping.

Despite major advances in next-generation sequencing, assembly of sequencing data, especially data from novel microorganisms or re-emerging pathogens, remains constrained by the lack of suitable reference sequences. De novo assembly is the best approach to achieve an accurate finished sequence, but multiple sequencing platforms or paired-end libraries are often required to achieve full genome coverage. In this study, we demonstrated a method to assemble complete bacterial genome sequences by integrating shotgun Roche 454 pyrosequencing with optical whole genome mapping (WGM). The whole genome restriction map (WGRM) was used as the reference to scaffold de novo assembled sequence contigs through a stepwise process. Large de novo contigs were placed in the correct order and orientation through alignment to the WGRM. De novo contigs that were not aligned to WGRM were merged into scaffolds using contig branching structure information. These extended scaffolds were then aligned to the WGRM to identify the overlaps to be eliminated and the gaps and mismatches to be resolved with unused contigs. The process was repeated until a sequence with full coverage and alignment with the whole genome map was achieved. Using this method we were able to achieved 100% WGRM coverage without a paired-end library. We assembled complete sequences for three distinct genetic components of a clinical isolate of Providencia stuartii: a bacterial chromosome, a novel bla NDM-1 plasmid, and a novel bacteriophage, without separately purifying them to homogeneity.

The Antimicrobial Resistance Monitoring and Research (ARMoR) Program: The US Department of Defense Response to Escalating Antimicrobial Resistance

Responding to escalating antimicrobial resistance (AMR), the US Department of Defense implemented an enterprise-wide collaboration, the Antimicrobial Resistance Monitoring and Research Program, to aid in infection prevention and control. It consists of a network of epidemiologists, bioinformaticists, microbiology researchers, policy makers, hospital-based infection preventionists, and healthcare providers who collaborate to collect relevant AMR data, conduct centralized molecular characterization, and use AMR characterization feedback to implement appropriate infection prevention and control measures and influence policy. A particularly concerning type of AMR, carbapenem-resistant Enterobacteriaceae, significantly declined after the program was launched. Similarly, there have been no further reports or outbreaks of another concerning type of AMR, colistin resistance in Acinetobacter, in the Department of Defense since the program was initiated. However, bacteria containing AMR-encoding genes are increasing. To update program stakeholders and other healthcare systems facing such challenges, we describe the processes and impact of the program.