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Klebsiella pneumoniae are a leading cause of healthcare-associated infections worldwide. In particular, strains expressing extended-spectrum β-lactamases (ESBLs) and carbapenemases pose serious treatment challenges, leading the World Health Organization (WHO) to designate ESBL and carbapenem-resistant Enterobacteriaceae as 'critical' threats to human health. Research efforts to combat these pathogens can be supported by accessibility to diverse and clinically relevant isolates for testing novel therapeutics. Here, we describe a panel of 100 diverse K. pneumoniae isolates that are publicly available to assist the research community in this endeavour. Whole-genome sequencing (WGS) was performed on 3878 K . pneumoniae clinical isolates housed at the Multidrug-Resistant Organism Repository and Surveillance Network. The isolates were cultured from 63 facilities in 19 countries between 2001 and 2020. Core-genome multilocus sequence typing and high-resolution single-nucleotide polymorphism-based phylogenetic analyses captured the genetic diversity of the collection and were used to select the final panel of 100 isolates. In addition to known multidrug-resistant (MDR) pandemic lineages, the final panel includes hypervirulent lineages and isolates with specific and diverse resistance genes and virulence biomarkers. A broad range of antibiotic susceptibilities, ranging from pan-sensitive to extensively drug-resistant isolates, are described. The panel collection, and all associated metadata and genome sequences, are available at no additional cost and will be an important resource for the research community and for the design and development of novel antimicrobial agents and diagnostics against this important pathogen.

Background: WHO has identified Marburg virus as an emerging virus requiring urgent vaccine research and development, particularly due to its recent emergence in Ghana. We report results from a first-in-human clinical trial evaluating a replication-deficient recombinant chimpanzee adenovirus type 3 (cAd3)-vectored vaccine encoding a wild-type Marburg virus Angola glycoprotein (cAd3-Marburg) in healthy adults., Methods: We did a first-in-human, phase 1, open-label, dose-escalation trial of the cAd3-Marburg vaccine at the Walter Reed Army Institute of Research Clinical Trials Center in the USA. Healthy adults aged 18-50 years were assigned to receive a single intramuscular dose of cAd3-Marburg vaccine at either 1 × 10 10 or 1 × 10 11 particle units (pu). Primary safety endpoints included reactogenicity assessed for the first 7 days and all adverse events assessed for 28 days after vaccination. Secondary immunogenicity endpoints were assessment of binding antibody responses and T-cell responses against the Marburg virus glycoprotein insert, and assessment of neutralising antibody responses against the cAd3 vector 4 weeks after vaccination. This study is registered with ClinicalTrials.gov, NCT03475056., Findings: Between Oct 9, 2018, and Jan 31, 2019, 40 healthy adults were enrolled and assigned to receive a single intramuscular dose of cAd3-Marburg vaccine at either 1 × 10 10 pu (n=20) or 1 × 10 11 pu (n=20). The cAd3-Marburg vaccine was safe, well tolerated, and immunogenic. All enrolled participants received cAd3-Marburg vaccine, with 37 (93%) participants completing follow-up visits; two (5%) participants moved from the area and one (3%) was lost to follow-up. No serious adverse events related to vaccination occurred. Mild to moderate reactogenicity was observed after vaccination, with symptoms of injection site pain and tenderness (27 [68%] of 40 participants), malaise (18 [45%] of 40 participants), headache (17 [43%] of 40 participants), and myalgia (14 [35%] of 40 participants) most commonly reported. Glycoprotein-specific antibodies were induced in 38 (95%) of 40 participants 4 weeks after vaccination, with geometric mean titres of 421 [95% CI 209-846] in the 1 × 10 10 pu group and 545 [276-1078] in the 1 × 10 11 pu group, and remained significantly elevated at 48 weeks compared with baseline titres (39 [95% CI 13-119] in the 1 ×10 10 pu group and 27 [95-156] in the 1 ×10 11 pu group; both p<0·0001). T-cell responses to the glycoprotein insert and neutralising responses against the cAd3 vector were also increased at 4 weeks after vaccination., Interpretation: This first-in-human trial of this cAd3-Marburg vaccine showed the agent is safe and immunogenic, with a safety profile similar to previously tested cAd3-vectored filovirus vaccines. 95% of participants produced a glycoprotein-specific antibody response at 4 weeks after a single vaccination, which remained in 70% of participants at 48 weeks. These findings represent a crucial step in the development of a vaccine for emergency deployment against a re-emerging pathogen that has recently expanded its reach to new regions., Funding: National Institutes of Health., Competing Interests: Declaration of interests NJS is listed on patents involving cAd3-vectored vaccines. All other authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objectives: Pseudomonas aeruginosa is a leading cause of community- and hospital-acquired infections. Successful treatment is hampered by its remarkable ability to rapidly develop resistance to antimicrobial agents, primarily through mutation. In response, WHO listed carbapenem-resistant P. aeruginosa as a Priority 1 (Critical) pathogen for research and development of new treatments. A key resource in developing effective countermeasures is access to diverse and clinically relevant strains for testing. Herein we describe a panel of 100 diverse P. aeruginosa strains to support this endeavour., Methods: WGS was performed on 3785 P. aeruginosa isolates in our repository. Isolates were cultured from clinical samples collected from healthcare facilities around the world between 2003 and 2017. Core-genome MLST and high-resolution SNP-based phylogenetic analyses were used to select a panel of 100 strains that captured the genetic diversity of this collection. Antibiotic susceptibility testing was also performed using 14 clinically relevant antibiotics., Results: This 100-strain diversity panel contained representative strains from 91 different STs, including genetically distinct strains from major epidemic clones ST-111, ST-235, ST-244 and ST-253. Seventy-one distinct antibiotic susceptibility profiles were identified ranging from pan-susceptible to pan-resistant. Known resistance alleles as well as the most prevalent mutations underlying the antibiotic susceptibilities were characterized for all isolates., Conclusions: This panel provides a diverse and comprehensive set of P. aeruginosa strains for use in developing solutions to antibiotic resistance. The isolates and available metadata, including genome sequences, are available to industry, academia, federal and other laboratories at no additional cost., (Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy 2021. This work is written by a US Government employee and is in the public domain in the US.)

Background: Recent deadly outbreaks of Marburg virus underscore the need for an effective vaccine. A summary of the latest research is needed for this WHO priority pathogen. This systematic review aimed to determine progress towards a vaccine for Marburg virus., Methods: Article search criteria were developed to query PubMed for peer-reviewed articles from 1990 through 2019 on Marburg virus vaccine clinical trials in humans and pre-clinical studies in non-human primates (NHP). Abstracts were reviewed by two authors. Relevant articles were reviewed in full. Discrepancies were resolved by a third author. Data abstracted included year, author, title, vaccine construct, number of subjects, efficacy, and demographics. Assessment for risk of bias was performed using the Syrcle tool for animal studies, and the Cochrane Collaboration risk of bias tool for human studies., Results: 101 articles were identified; 27 were related to Marburg vaccines. After full text review, 21 articles were selected. 215 human subjects were in three phase 1 clinical trials, and 203 NHP in 18 studies. Vaccine constructs were DNA plasmids, recombinant vesicular stomatitis virus (VSV) vectors, adenovirus vectors, virus-like particles (VLP), among others. Two human phase 1 studies of DNA vaccines had 4 adverse effects requiring vaccine discontinuation among 128 participants and 31-80% immunogenicity. In NHP challenge studies, 100% survival was seen in 6 VSV vectored vaccines, 2 DNA vaccines, 2 VLP vaccines, and in 1 adenoviral vectored vaccine., Conclusion: In human trials, two Marburg DNA vaccines provided either low immunogenicity or a failure to elicit durable immunity. A variety of NHP candidate Marburg vaccines demonstrated favorable survival and immunogenicity parameters, to include VSV, VLP, and adenoviral vectored vaccines. Elevated binding antibodies appeared to be consistently associated with protection across the NHP challenge studies. Further human trials are needed to advance vaccines to limit the spread of this highly lethal virus., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Over the past two decades, Acinetobacter baumannii has emerged as a leading cause of nosocomial infections worldwide. Of particular concern are panresistant strains, leading the World Health Organization (WHO) to designate carbapenem-resistant A. baumannii as a priority 1 (critical) pathogen for research and development of new antibiotics. A key component in supporting this effort is accessibility to diverse and clinically relevant strains for testing. Here, we describe a panel of 100 diverse A. baumannii strains for use in this endeavor. Whole-genome sequencing was performed on 3,505 A. baumannii isolates housed at the Multidrug-Resistant Organism Repository and Surveillance Network. Isolates were cultured from clinical samples at health care facilities around the world between 2001 and 2017. Core-genome multilocus sequence typing and high-resolution single nucleotide polymorphism (SNP)-based phylogenetic analyses were used to select a final panel of 100 strains that captured the genetic diversity of the collection. Comprehensive antibiotic susceptibility testing was also performed on all 100 isolates using 14 clinically relevant antibiotics. The final 100-strain diversity panel contained representative strains from 70 different traditional Pasteur scheme multilocus sequence types, including major epidemic clones. This diversity was also reflected in antibiotic susceptibility and antimicrobial resistance (AMR) gene content, with phenotypes ranging from pansensitive to panresistant, and over 100 distinct AMR gene alleles identified from 32 gene families. This panel provides the most diverse and comprehensive set of A. baumannii strains for use in developing solutions for combating antibiotic resistance. The panel and all available metadata, including genome sequences, will be available to industry and academic institutions and federal and other laboratories free of charge.

Background: Governments and health care regulators now require hospitals and nursing homes to establish programs to monitor and report antimicrobial consumption and resistance. However, additional resources were not provided. We sought to develop an approach for monitoring antimicrobial resistance and consumption that health care systems can implement with minimal added costs or modifications to existing diagnostic and informatics infrastructure., Methods: Using (1) the electronic laboratory information system of a nationwide managed care network, (2) the 3 most widely used commercial microbiology diagnostic platforms, and (3) Staphylococcus aureus , one of the most common causes of infections worldwide, as a prototype, we validated the approach dubbed "SAVANT" for Semi-Automated Visualization and ANalysis of Trends. SAVANT leverages 3 analytical methods (time series analysis, the autoregressive integrated moving average, and generalized linear regression) on either commercial or open source software to report trends in antistaphylococcal use and resistance., Results: All laboratory results from January 2010 through December 2015 from an annual average of 9.2 million health care beneficiaries were queried. Inpatient and outpatient prescription rates were calculated for 8 key antistaphylococcal compounds. Trends and relationships of antistaphylococcal consumption and resistance among 81 840 unique S. aureus isolates from >6.5 million cultures were revealed., Conclusions: Using existing or freely available resources, SAVANT was successfully implemented across a complex and geographically dispersed 280-hospital network, bridging a critical gap between medical informatics, large-scale data analytics, and mandatory reporting of health care quality metrics.

The loss of fitness in colistin-resistant (CR) Acinetobacter baumannii was investigated using longitudinal isolates from the same patient. Early CR isolates were outcompeted by late CR isolates for growth in broth and survival in the lungs of mice. Fitness loss was associated with an increased susceptibility to oxidative stress since early CR strains had reduced in vitro survival in the presence of hydrogen peroxide and decreased catalase activity compared to that of late CR and colistin-susceptible (CS) strains., (Copyright © 2017 Jones et al.)

Background: Three full doses of RTS,S/AS01 malaria vaccine provides partial protection against controlled human malaria parasite infection (CHMI) and natural exposure. Immunization regimens, including a delayed fractional third dose, were assessed for potential increased protection against malaria and immunologic responses., Methods: In a phase 2a, controlled, open-label, study of healthy malaria-naive adults, 16 subjects vaccinated with a 0-, 1-, and 2-month full-dose regimen (012M) and 30 subjects who received a 0-, 1-, and 7-month regimen, including a fractional third dose (Fx017M), underwent CHMI 3 weeks after the last dose. Plasmablast heavy and light chain immunoglobulin messenger RNA sequencing and antibody avidity were evaluated. Protection against repeat CHMI was evaluated after 8 months., Results: A total of 26 of 30 subjects in the Fx017M group (vaccine efficacy [VE], 86.7% [95% confidence interval [CI], 66.8%-94.6%]; P < .0001) and 10 of 16 in the 012M group (VE, 62.5% [95% CI, 29.4%-80.1%]; P = .0009) were protected against infection, and protection differed between schedules (P = .040, by the log rank test). The fractional dose boosting increased antibody somatic hypermutation and avidity and sustained high protection upon rechallenge., Discussions: A delayed third fractional vaccine dose improved immunogenicity and protection against infection. Optimization of the RTS,S/AS01 immunization regimen may lead to improved approaches against malaria., Clinical Trials Registration: NCT01857869., (Published by Oxford University Press on behalf of the Infectious Diseases Society of America, 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

During a military public health laboratory symposium held in 1999, concerns were raised that the military health system lacked a standardized antimicrobial resistance (AMR) surveillance system that allowed comparison of data across sites, investigation of trends, and understanding of resistance mechanisms. The purpose of this review was to assess if current AMR activities in the military health system have addressed the aforementioned gaps. It was determined that much progress has already been made within the Department of Defense with respect to monitoring and understanding AMR through initiatives such as the Antimicrobial Resistance Monitoring and Research Program-a strong Department of Defense-wide surveillance program. These surveillance efforts can be made more robust through harmonization of testing and reporting structures across military treatment facilities, and by encouraging military treatment facility participation., (Reprint & Copyright © 2015 Association of Military Surgeons of the U.S.)

Whether carbapenem or fluoroquinolone usage is correlated with carbapenem-resistant Enterobacteriaceae (CRE) has not been investigated at the level of an entire US nationwide managed health care system. We analyzed 75 million person-years of surveillance and 1,969,315 cultures from all 266 hospitals in the geographically dispersed US military health system. Incidences of CRE remained under 1 case per 100,000 person-years. Incidences of CRE increased relative to 2005 baseline levels in 3 of 7 subsequent years, then decreased in 2012 (P<0.05). Incident proportions of carbapenem resistance (CR) differed significantly among years, geographical regions, and bacterial species. Although use and resistance strongly correlated (R>0.80) for several "drug-bug" combinations, none were significant at the national or facility level. One exception was that inpatient consumption of fluoroquinolones was significantly correlated (P=0.0007) with CR in Escherichia coli when data from the major referral centers of the Southern and Northern regions were combined., (Published by Elsevier Inc.)

A 22-year-old male developed a recurrent sacral abscess associated with embedded shrapnel following a blast injury. Cultures grew extended-spectrum β-lactamase (ESBL)-producing, carbapenem-susceptible Escherichia coli. Ertapenem was administered, but the infection recurred after each course of antibiotics. Initial surgical interventions were unsuccessful, and subsequent cultures yielded E. coli and Morganella morganii, both nonsusceptible to carbapenems. The isolates were Carba NP test negative, gave ambiguous results with the modified Hodge test, and amplified the bla(OXA48)-like gene by real-time PCR. All E. coli isolates were sequence type 131 (ST131), carried nine resistance genes (including bla(CTX-M-27)) on an IncF plasmid, and were identical by genome sequencing, except for 150 kb of plasmid DNA in carbapenem-nonsusceptible isolates only. Sixty kilobases of this was shared by M. morganii and represented an IncN plasmid harboring bla(OXA-181). In M. morganii, the gene was flanked by IS3000 and ISKpn19, but in all but one of the E. coli isolates containing bla(OXA-181), a second copy of ISKpn19 had inserted adjacent to IS3000. To the best of our knowledge, this is the first report of bla(OXA-181) in the virulent ST131 clonal group and carried by the promiscuous IncN family of plasmids. The tendency of M. morganii to have high MICs of imipenem, a bla(OXA-181) substrate profile that includes penicillins but not extended-spectrum cephalosporins, and weak carbapenemase activity almost resulted in the presence of bla(OXA-181) being overlooked. We highlight the importance of surveillance for carbapenem resistance in all species, even those with intrinsic resistances, and the value of advanced molecular techniques in detecting subtle genetic changes., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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., (Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Unlabelled: 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., (Copyright © 2014 Jacobs et al.)

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.

We describe a real-time PCR-based assay capable of simultaneously detecting femA (Staphylococcus aureus-specific), mecA (methicillin resistance), qacA/B (chlorhexidine tolerance), and mupA (high-level mupirocin resistance) from bacterial cells in less than 90 minutes. The assay was validated with 1968 clinical MRSA submitted to a surveillance network., (© 2013.)

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.

A carbapenem-resistant Acinetobacter baumannii strain was isolated from the peritoneal fluid of a patient with complicated intra-abdominal infection and evaluated at the Multidrug-resistant Organism Repository and Surveillance Network by whole-genome sequencing and real-time PCR. The isolate was sequence type 25 and susceptible to colistin and minocycline, with low MICs of tigecycline. blaNDM-1 was located on a plasmid with >99% homology to pNDM-BJ02. The isolate carried numerous other antibiotic resistance genes, including the 16S methylase gene, armA.

A carbapenem-resistant Alcaligenes faecalis strain was isolated from a surveillance swab of a service member injured in Afghanistan. The isolate was positive for bla(NDM) by real-time PCR. Species identification was reevaluated on three identification systems but was inconclusive. Genome sequencing indicated that the closest relative was Acinetobacter schindleri and that bla(NDM-1) was carried on a plasmid that shared >99% identity with one identified in an Acinetobacter lwoffii isolate. The isolate also carried a novel chromosomally encoded class D oxacillinase.

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.

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 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 blaKPC and blaNDM, two of the most important carbapenemases, directly from culture in less than 90 min. The assay was validated with blaKPC- and blaNDM-carrying clinical isolates and demonstrated 100% concordance with the Carba NP test.

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.

In response to global concerns over the spread of the New Delhi metallo-β-lactamase gene 1, bla(NDM-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 bla(NDM-1). Both strains were identical by pulsed-field gel electrophoresis (PFGE). bla(NDM-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 bla(NDM-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 bla(OXA-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 bla(NDM-1) gene further highlights the potential mobility associated with this gene.

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.

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.

Background: Current US military clinical practice guidelines permit emergency transfusions of non-Food and Drug Administration (FDA)-compliant freshly collected blood products in theaters of war. This investigation aimed to characterize the risks of transfusion-transmitted infections (TTIs) associated with battlefield transfusions of non-FDA-compliant blood products., Study Design and Methods: US Service members who received emergency transfusion products in Iraq and Afghanistan (March 1, 2002-September 30, 2007) were tested for hepatitis C virus (HCV), human immunodeficiency virus (HIV), and hepatitis B virus (HBV) infections using reposed pre- and posttransfusion sera. Selected regions of viral genomes from epidemiologically linked infected recipients and their donors were sequenced and compared., Results: Of 761 US Service members who received emergency transfusion products, 475 were tested for HCV, 472 for HIV, and 469 for HBV. One transfusion-transmitted HCV infection (incidence rate of 2.1/1000 persons) was identified. The pretransfusion numbers (prevalence per 1000 persons) were HCV-four (8/1000), HIV-zero (0/1000), chronic HBV-two (4 /1000), and naturally immune (antibody to HBV core antigen)-nine (19/1000)., Conclusion: One HCV TTI was determined to be associated with emergency blood product use. The pretransfusion HCV and HBV prevalence in transfusion recipients, themselves members of the potential donor population, indicates better characterization of the deployed force's actual donor population, and further investigations of the TTI prevalence in these donors are needed. These data will inform countermeasure development and clinical decision making., (© 2010 American Association of Blood Banks.)