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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 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.

Background Malaria remains the top infectious disease threat facing the U.S. military in many forward operating environments. Compliance with malaria chemoprophylaxis remains a critical component in preventing malaria in the deployed Service Member. Studies of previous military operations show that compliance is consistently higher with weekly versus daily dosing regimens. Current FDA approved weekly chemoprophylaxis options have contraindications that can limit prescribing. The combination of chloroquine (CQ) with azithromycin (AZ) has previously been shown to be an efficacious treatment option for malaria, has pharmacokinetics compatible with weekly dosing, and has shown synergy when combined in vitro. Methods In this open label study, 18 healthy volunteers, aged 18–50 years (inclusive), were randomly assigned to receive either 300 mg CQ or 300 mg CQ and 2 gm azithromycin (CQAZ) of directly observed therapy, weekly for 3 weeks prior to undergoing mosquito bite challenge with chloroquine-resistant Plasmodium falciparum. Volunteers that remained asymptomatic and had no evidence of parasitaemia continued to receive weekly post-exposure chemoprophylaxis for 3 weeks following malaria challenge. The primary endpoint was the number of volunteers that remained asymptomatic and had no evidence of parasitaemia 28 days after the malaria challenge. Results All 6 (100%) volunteers randomized to the CQ control group became symptomatic with parasitaemia during the 28-day post-challenge period. Only 1/12 (8.3%) of volunteers in the CQAZ group developed symptoms and parasitaemia during the 28-day post-challenge period. However, after chemoprophylaxis was discontinued an additional 6 volunteers developed parasitaemia between days 28–41 after challenge, with 4 of 6 experiencing symptoms. 80% of subjects in the CQAZ group experienced treatment related gastrointestinal adverse events (including 13% that experienced severe nausea) compared to 38% in the CQ group. A comparison of the pharmacokinetics in the CQAZ group demonstrated higher azithromycin Cmax (p = 0.03) and AUC (p = 0.044) levels in those volunteers who never became parasitaemic compared to those who did. Conclusion Given the high rate of side effects and poor efficacy when administered for 3 weeks before and after challenge, the combination of weekly chloroquine and azithromycin is a suboptimal regimen combination for weekly malaria chemoprophylaxis. Trial registration ClinicalTrials.gov NCT03278808

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.

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 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.

Rapidly growing nontuberculous mycobacteria (RGNTM) have yet to be described in combat-related injuries. This study investigates the epidemiology, clinical findings, treatment, and outcomes of RGNTM infections among combat casualties wounded in Afghanistan from 2010 to 2012.Patients with RGNTM were identified from the Department of Defense Trauma Registry through the Trauma Infectious Disease Outcomes Study. Trauma history, surgical management, and clinical data were collected. Six isolates from patients requiring antimycobacterial therapy were sequenced.Seventeen cases were identified. Six cases, predominantly associated with Mycobacterium abscessus, required aggressive debridement and a median of 180 days of multidrug antimycobacterial therapy that included clofazimine. M. abscessus isolates expressed the erythromycin resistance methylase (erm(41)) gene for inducible macrolide resistance, yet there were no clinical treatment failures when macrolides were utilized in combination therapy. No clonal similarity between M. abscessus isolates was found. Eleven cases had positive wound cultures, but did not require antimycobacterial therapy. The median duration of time of injury to first detection of a RGNTM was 57 days.This represents the first report of RGNTM infections in war-wounded patients. RGNTM should be recognized as potential pathogens in grossly infected combat wounds. Surgical debridement and multidrug antimycobacterial therapy, when clinically indicated, was associated with satisfactory clinical outcomes.

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 IS 3000 and IS Kpn19 , but in all but one of the E. coli isolates containing bla OXA-181 , a second copy of IS Kpn19 had inserted adjacent to IS 3000 . 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.

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.

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.

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.

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 IS 5 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 .

Awareness, responsiveness, and throughput characterize an approach for enhancing the clinical impact of whole genome sequencing for austere environments and for large geographically dispersed health systems. This Department of Defense approach is informing interagency efforts linking antibiograms of multidrug-resistant organisms to their genome sequences in a public database.

Carbapenem-resistant Pseudomonas aeruginosa, Acinetobacter spp., and Enterobacteriaceae pose urgent public health threats. The differential burden, relative risks, associations with antimicrobial consumption, and temporal trends of those taxa in large, geographically diverse U.S. health systems remain under reported. Electronic records of all patients in a geographically dispersed 280-hospital managed-care system from 2005 to 2014 were reviewed. Carbapenem-resistant strains were identified based on Clinical and Laboratory Standards Institute guidelines and breakpoints. A total of 360,000 potentially carbapenem-resistant strains were identified from 14.7 million cultures (80% infecting and 20% surveillance). Isolation of bacteria overseas or isolation from the bloodstream was associated with a higher relative risks of carbapenem resistance (CR; P < 0.0001). Enterobacteriaceae were isolated 11 times more frequently than P. aeruginosa and Acinetobacter spp. However, compared to Enterobacteriaceae , the CR levels were 73-fold and 210-fold higher in P. aeruginosa and Acinetobacter spp., respectively. Significant differences in the relative risk of CR between taxa, anatomic, and geographic locations persisted after adjustment for other variables, the biggest differences occurring between taxa. Overall, CR rates increased for Enterobacteriaceae ( P = 0.03) and decreased for Acinetobacter spp. and P. aeruginosa ( P < 0.0001). These data provide a useful baseline for resistance trending and have implications for surveillance. Infections acquired overseas and bloodstream infections are particularly important areas for continued monitoring.

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.

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. bla NDM-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 .

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.

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.

Responding to escalating antimicrobial resistance (AMR), the US Department of Defense implemented an en-terprise-widecollaboration,theAntimicrobialResistanceMonitoringandResearchProgram,toaidininfectionpreventionandcontrol.Itconsistsofanetworkofepidemiologists,bioinformaticists,microbiologyresearchers,policy makers, hospital-based infection preventionists, and healthcare providers who collaborate to collect rel-evant AMR data, conduct centralized molecular characterization, and use AMR characterization feedback toimplementappropriateinfectionpreventionandcontrolmeasuresandinfluencepolicy.Aparticularlyconcern-ing type of AMR, carbapenem-resistant Enterobacteriaceae, significantly declined after the program waslaunched. Similarly, there have been no further reports or outbreaks of another concerning type of AMR, co-listin resistance in Acinetobacter, in the Department of Defense since the program was initiated. However, bac-teria containing AMR-encoding genes are increasing. To update program stakeholders and other healthcaresystems facing such challenges, we describe the processes and impact of the program.Keywords. antimicrobial resistance; surveillance; infection prevention; Department of Defense.The US response to escalating antimicrobial resistance(AMR) has been described as sluggish and more than adecade behind national organizations elsewhere, suchas the European Union [1]. In an effort to close thatgap and provide safe, high quality care, the US Depart-mentofDefense(DoD)launchedtheAntimicrobialRe-sistance Monitoring and Research (ARMoR) Programin 2009. It consists of an integrated network of policy-making and hospital-based infection preventionists,individual healthcare providers, research microbiolo-gists and bioinformaticists, epidemiologists, and seniorhealthcare leaders. The program is government fundedand thus includes all US taxpayers as stakeholders. Toupdatethese stakeholders,and because other healthcaresystems may find part or all of the approach applicabletotheirefforts, wedescribethe program’s processes andhighlight its impact.METHODS

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 (P0.05). Incident proportions of carbapenem resistance (CR) differed significantly among years, geographical regions, and bacterial species. Although use and resistance strongly correlated (R0.80) 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.

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.

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.

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.

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.

The prevalence of multidrug-resistant infections in personnel wounded in Iraq and Afghanistan has made it challenging for physicians to choose effective therapeutics in a timely fashion. To address the challenge of identifying the potential for drug resistance, we have developed the Antimicrobial Resistance Determinant Microarray (ARDM) to provide DNAbased analysis for over 250 resistance genes covering 12 classes of antibiotics. Over 70 drug-resistant bacteria from different geographic regions have been analyzed on ARDM, with significant differences in patterns of resistance identified: genes for resistance to sulfonamides, trimethoprim, chloramphenicol, rifampin, and macrolide-lincosamidesulfonamide drugs were more frequently identified in isolates from sources in Iraq/Afghanistan. Of particular concern was the presence of genes responsible for resistance to many of the last-resort antibiotics used to treat war traumaassociated infections.© (2012) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.

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.

Objectives To investigate the epidemiology and risk factors of gonorrhoea (GC) or chlamydia (CT) coinfection in an HIV-positive US military cohort, focusing on the time after participants9 knowledge of HIV diagnosis. Methods The authors analysed data from 4461 participants enrolled in the US Military Natural History Study cohort for GC or CT infection ≥6 months after their HIV-positive test. Results During a mean follow-up of 7.08 years, 482 (11%) participants acquired a GC or CT infection. Of these, 283 (6%) acquired a GC infection, 278 (6%) acquired a CT infection and 123 (3%) had multiple GC or CT infections during follow-up. Risk of GC or CT infection was significantly greater in those younger, male, African–American and with a history of GC or CT infection. Conclusions Frequent GC and CT diagnoses observed among members of this HIV-positive cohort indicate substantial ongoing risk behaviours that raise concerns for HIV transmission and underscore the need for continued screening to help identify and treat these sexually transmitted infections in this population.

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.

Accidents or violence can result in penetrating trauma in the adult population. Contaminated penetrating foreign bodies introduced at the time of wounding cause infection, especially high velocity projectiles, which result in cavitation. Surgical debridement reduces potential infection; however, perioperative antibiotics are usually indicated owing to studies demonstrating high rates of sepsis in the pre-antibiotic era. Trauma-associated pathogens include Gram-positive, Gram-negative and anaerobic pathogens. Antibiotic resistance is increasing, and several recent panels have sought to develop guidelines for perioperative prevention and empiric treatment of infection to limit usage and reduce selective pressure for resistance. We review infections of the CNS, thorax, abdomen and extremities following penetrating trauma injury, as well as the data supporting a reasonable antimicrobial approach.

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.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.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).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.

War-trauma, especially due to blast injury, can be associated with long bone fracture. Immediate external fixation of fractures, followed by internal fixation when the patient is medically stabilized (damage control orthopedics [DCO]), is the U.S. Army policy for war-related fractures. Data on infectious outcomes when DCO is used for war-trauma fractures are scant.A retrospective review of U.S. war-trauma patients from 2003 to 2007 with femoral or tibial fractures treated by DCO was conducted. Fisher's Exact and Mann-Whitney tests were used for comparisons.Fifty-eight soldiers were identified. Fifty-five were males with a median age of 26 years (19-54 years) and a median time to internal fixation by intramedually nailing of 9 days (4-414 days). Eighty-eight percent of fractures were open, and 57% were femoral fractures. The median duration of follow-up was 447 days (20-1,340 days). Fracture site infection occurred in 40% (23 of 58), with suspected osteomyelitis in 17% (10 of 58). Of infected nails, fracture union occurred in 70% and nail retention in 57%. Median time to infection after nail placement was 15 days (0-717 days) with 75% of infections occurring by day 113. Multiple bacterial pathogens including Acinetobacter baumannii and Staphylococcus spp. were causative organisms. Blast injuries occurred in 91% of infected versus 47% of uninfected (p = 0.005). There was no difference between infections occurring in femoral (61%) versus tibial (39%) (p = 0.620) location.Infection was associated with 40% of DCO-associated intramedullary nails. Blast injury was a predictor of infection. Despite infection, fracture union and nail retention rates were high, suggesting a good outcome.

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.

To evaluate the impact of fluconazole prophylaxis on the incidence of fungal infections and on mortality among critically ill surgical patients.Meta-analysis of randomized, placebo-controlled trials of fluconazole prophylaxis.Subjects participating in the clinical trials in this area.We identified four randomized studies comparing fluconazole to placebo for prevention of fungal infections in the surgical intensive care unit (SICU). The studies enrolled 626 patients and used differing dosing regimens of fluconazole. All trials were double-blind and two were multicenter studies. Fluconazole administration significantly reduced the incidence of fungal infections (pooled odds ratio, 0.44; 95% confidence interval, 0.27-0.72; p.001). However, fluconazole prophylaxis was not associated with a survival advantage (pooled OR for mortality, 0.87; 95% confidence interval, 0.59-1.28; p = NS). Fluconazole did not statistically alter the rate of candidemia, as this was low across the studies and developed in only 2.2% of all participants. Performing a sensitivity analysis and including two additional studies that indirectly examined fluconazole prophylaxis in the critically ill did not change our observations. Data from the reports reviewed were insufficient to allow comment on the impact of fluconazole prophylaxis on resource utilization, the distribution of non-albicans species of Candida, and the emergence of antifungal resistance. Generally, fluconazole appeared to be safe for SICU patients.Prophylactic fluconazole administration for prevention of mycoses in SICU patients appears to successfully decrease the rate of these infections, but this strategy does not improve survival. The absence of a survival advantage may reflect the few studies in this area and the possibility that this issue has not been adequately studied. Because of the potential for both resistance and emergence of non-albicans isolates, clinicians must consider these issues when evaluating fluconazole prophylaxis in the SICU. Future trials should focus on more precisely identifying patients at high risk for fungal infections and on determining if broader use of fluconazole alters the distribution of candidal species seen in the SICU and impacts measures of resource utilization such as length of stay and duration of mechanical ventilation.