Fatma Onmus-Leone

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

BACKGROUNDnColistin 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.nnnMETHODSnUnder a quality-improvement directive to intensify infection-control measures, extremely drug-resistant (XDR) bacteria undergo phenotypic and molecular analysis.nnnRESULTSnTwenty-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.nnnCONCLUSIONSnWe 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.

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.

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

BACKGROUNDnSevere Acinetobacter baumannii infections in immunocompetent patients are uncommon, and the virulence mechanisms of this organism are not fully understood.nnnMETHODSnFollowing 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.nnnRESULTSnTwo 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.nnnCONCLUSIONSnClade 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.

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.

A multidrug-resistance surveillance network: 1 year on

www.thelancet.com/infection Vol 12 August 2012 587 caused by MRSA and A baumannii, and fungal wound infections. Timely feedback sped up identifi cation of the source, enhanced disinfection and implementation of transmissionbased precautions, and increased antibiotic stewardship. We developed and validated multiplex real-time PCR platforms to test isolates for the presence of mupA, qacA/B, LG251, all variants of blaNDM and blaKPC, and the most common variants of blaVIM. blaNDM-1 carried on a novel plasmid was detected in Providencia stuartii from Afghanistan. We were fi rst to report qacA/B in MRSA in USA, which had higher tolerance of chlorhexidine gluconate than those that did not have the gene. A cluster of colistin-resistant organisms emerged during therapy with colistin. We combined optical mapping and sequencing to identify gene copy number changes in sequential Acinetobacter isolates from the same patient. We improved the speed and reduced the cost of optical genome mapping by successfully mapping multiple genomes on the standard map card. MRSN also does canine and environmental surveillance. We thank our current partners and invite new collaborators.

Complete Genome Sequence of Providencia stuartii Clinical Isolate MRSN 2154

Here we present the complete genome sequence of Providencia stuartii MRSN 2154, isolated from an Afghan national. P. stuartii is a Gram-negative bacillus capable of causing infections in a wide variety of human tissues. Because Providencia readily acquires plasmids bearing drug resistance loci, it is of growing clinical significance.

The Challenges of Implementing Next Generation Sequencing Across a Large Healthcare System, and the Molecular Epidemiology and Antibiotic Susceptibilities of Carbapenemase-Producing Bacteria in the Healthcare System of the U.S. Department of Defense

Objective We sought to: 1) provide an overview of the genomic epidemiology of an extensive collection of carbapenemase-producing bacteria (CPB) collected in the U.S. Department of Defense health system; 2) increase awareness of the public availability of the sequences, isolates, and customized antimicrobial resistance database of that system; and 3) illustrate challenges and offer mitigations for implementing next generation sequencing (NGS) across large health systems. Design Prospective surveillance and system-wide implementation of NGS. Setting 288-hospital healthcare network. Methods All phenotypically carbapenem resistant bacteria underwent CarbaNP® testing and PCR, followed by NGS. Commercial (Newbler and Geneious), on-line (ResFinder), and open-source software (Btrim, FLASh, Bowtie2, an Samtools) were used for assembly, SNP detection and clustering. Laboratory capacity, throughput, and response time were assessed. Results From 2009 through 2015, 27,000 multidrug-resistant Gram-negative isolates were submitted. 225 contained carbapenemase-encoding genes (most commonly blaKPC, blaNDM, and blaOXA23). These were found in 15 species from 146 inpatients in 19 facilities. Genetically related CPB were found in more than one hospital. Other clusters or outbreaks were not clonal and involved genetically related plasmids, while some involved several unrelated plasmids. Relatedness depended on the clustering algorithm used. Transmission patterns of plasmids and other mobile genetic elements could not be determined without ultra-long read, single-molecule real-time sequencing. 80% of carbapenem-resistant phenotypes retained susceptibility to aminoglycosides, and 70% retained susceptibility to fluoroquinolones. However, among the CPB-confirmed genotypes, fewer than 25% retained susceptibility to aminoglycosides or fluoroquinolones. Conclusion Although NGS is increasingly acclaimed to revolutionize clinical practice, resource-constrained environments, large or geographically dispersed healthcare networks, and military or government-funded public health laboratories are likely to encounter constraints and challenges as they implement NGS across their health systems. These include lack of standardized definitions and quality control metrics, limitations of short-read sequencing, insufficient bandwidth, and the current limited availability of very expensive and scarcely available sequencing platforms. Possible solutions and mitigations are also proposed.

Whole-Genome Analysis of Bartonellaancashensis, a Novel Pathogen Causing Verruga Peruana, Rural Ancash Region, Peru

The genus Bartonella contains >40 species, and an increasing number of these Bartonella species are being implicated in human disease. One such pathogen is Bartonella ancashensis, which was isolated in blood samples from 2 patients living in Caraz, Peru, during a clinical trial of treatment for bartonellosis. Three B. ancashensis strains were analyzed by using whole-genome restriction mapping and high-throughput pyrosequencing. Genome-wide comparative analysis of Bartonella species showed that B. ancashensis has features seen in modern and ancient lineages of Bartonella species and is more related to B. bacilliformis. The divergence between B. ancashensis and B. bacilliformis is much greater than what is seen between known Bartonella genetic lineages. In addition, B. ancashensis contains type IV secretion system proteins, which are not present in B. bacilliformis. Whole-genome analysis indicates that B. ancashensis might represent a distinct Bartonella lineage phylogenetically related to B. bacilliformis.

Relationships among cleaning, environmental DNA, and healthcare-associated infections in a new evidence-based design hospital.

OBJECTIVEnHospital environments influence healthcare-associated infection (HAI) patterns, but the role of evidenced-based design (EBD) and residual bacterial DNA (previously thought to be clinically inert) remain incompletely understood.nnnMETHODSnIn a newly built EBD hospital, we used culture-based and culture-free (molecular) assays, pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing (WGS) to determine: (1) patterns of environmental contamination with target organisms (TOs) and multidrug-resistant (MDR) target organisms (MDR-TOs); (2) genetic relatedness between environmentally isolated MDR-TO and those from HAIs; and (3) correlation between surface contamination and HAIs.nnnRESULTSnA total of 1,273 high-touch surfaces were swabbed before and after terminal cleaning during 77 room visits. Of the 2,546 paired swabs, 47% had cultivable biomaterial and 42% had PCR-amplifiable DNA. The ratios of TOs detected to surfaces assayed were 85 per 1,273 for the culture-based method and 106 per 1,273 for the PCR-based method. Sinks, toilet rails, and bedside tables most frequently harbored biomaterial. Although cleaned surfaces were less likely to have cultivable TOs than precleaned surfaces, they were not less likely to harbor bacterial DNA. The rate of MDR-TOs to surfaces swabbed was 0.1% (3/2546). Although environmental MDR-TOs and MDR-TOs from HAIs were genetically related by PFGE, WGS revealed that they were unrelated. Environmental levels of cultivable Enterococcus spp. and E. coli DNA were positively correlated with infection incidences (P<.04 and P<.005, respectively).nnnCONCLUSIONnMDR-TOs were rarely detected during surveillance and were not implicated in HAIs. The roles of environmental DNA and EBD, particularly with respect to water-associated fixtures or the potential suppression of cultivable environmental MDR-TOs, warrant multicenter investigations.