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The potential use of Bacillus anthracis as a bioterrorism weapon requires a safe and effective vaccine that can be immediately distributed for mass vaccination. Protective antigen (PA), a principal component of virulence factors edema toxin and lethal toxin of B. anthracis, has been the topic of extensive research. Previously, full-length PA (PA83) was manufactured using a transient plant-based expression system. Immunization with this PA83 antigen formulated with Alhydrogel® adjuvant elicited strong neutralizing immune responses in mice and rabbits and protected 100% of rabbits from a lethal aerosolized B. anthracis challenge. This Phase 1 study evaluates this vaccine's safety and immunogenicity in healthy human volunteers.This first-in-human, single-blind, Phase 1 study was performed at a single center to investigate the safety, reactogenicity, and immunogenicity of the plant-derived PA83-FhCMB vaccine at four escalating dose levels (12.5, 25, 50 or 100 µg) with Alhydrogel® in healthy adults 18-49 years of age (inclusive). Recipients received three doses of vaccine intramuscularly at 28-day intervals. Safety was evaluated on days 3, 7, and 14 following vaccination. Immunogenicity was assessed using an enzyme-linked immunosorbent assay (ELISA) and a toxin neutralizing antibody (TNA) assay on days 0, 14, 28, 56, 84, and 180.All four-dose ranges were safe and immunogenic, with no related serious adverse events observed. Peak ELISA Geometric Mean Concentration (GMC) and TNA EDThis is the first study demonstrating a plant-derived subunit anthrax vaccine's safety and immunogenicity in healthy adults. The results support further clinical investigation of the PA83-FhCMB vaccine. ClinicalTrials.gov identifier. NCT02239172.

Carbapenem-resistant Enterobacterales pose an urgent threat to human health worldwide. Klebsiella pneumoniae sequence type (ST) 14, initially identified in the Middle East and South-Asia and co-harbouring the carbapenemase genes bla OXA-232 and bla NDM-1, is now emerging globally. One such strain was detected in the USA in 2013 from a patient initially treated in India that also carried armA, a 16S rRNA methyltransferase that confers resistance to all clinically relevant aminoglycosides. Genetic and phenotypic changes were observed in 14 serial isolates collected from this chronically infected patient. The index isolate carried five plasmids, including an IncFIB–IncHI1B (harbouring armA and bla NDM-1), an IncFIA (bla CTX-M-15) and a ColE-like (bla OXA-232), and was extensively resistant to antibiotics. Four years later, a subsequent isolate had accumulated 34 variants, including a loss-of-function mutation in romA, resulting in tigecycline non-susceptibility. Importantly, this isolate now only carried two plasmids, including a large mosaic molecule made of fragments, all harbouring distinct toxin–antitoxin systems, from three of the canonical plasmids. Of the original acquired antibiotic resistance genes, this isolate only retained bla CTX-M-15, and as a result susceptibility to the carbapenems and amikacin was restored. Long-read sequencing of a subset of five representative isolates, collected between 2013 and 2017, allowed for the elucidation of the complex plasmid patterns and revealed the role of IS26-mediated plasmid reshuffling in the evolution of this clone. Such investigations of the mechanisms underlying plasmid stability, together with global and local surveillance programmes, are key to a better understanding of plasmid host range and dissemination.

Carbapenem-resistant

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 Plasmodium vivax controlled human malaria infection (PvCHMI) is an important tool for evaluation of drugs, vaccines, and pathologies associated with this parasite. However, there are few data on safety due to limited numbers of PvCHMIs performed. Methods We report clinical and laboratory data, including hematological and biochemical profiles and adverse events (AEs), following mosquito bite-induced PvCHMI in malaria-naive study participants. Malaria diagnosis and treatment initiation was based on microscopic analysis of Giemsa-stained slides. Exploratory molecular assays were used to detect parasites using real-time polymerase chain reaction (PCR). Results AEs were mild to moderate and no study-related severe AEs were observed in any study participants. The majority of symptoms were transient, resolving within 48 hours. Molecular diagnostic methods detected parasitemia in 100% of study participants before malaria diagnosis using microscopy. Of reported AEs, microscopy detected 67%–100%, quantitative PCR 79%–100%, and quantitative real-time reverse-transcription PCR 96%–100% of study participants prior to appearance of symptoms. Almost all symptoms appeared after initiation of treatment, likely as known consequence of drug treatment. Conclusions PvCHMI is safe with the majority of infections being detected prior to appearance of clinical symptoms, which can be further alleviated by using sensitive molecular methods for clinical diagnosis. Clinical Trials Registration. NCT01157897.

A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.1.345 variant carrying the E484K mutation was detected in 4 patients with no apparent epidemiological association from a hospital network in upstate New York. Subsequent analysis identified an additional 11 B.1.1.345 variants from this region between December 2020 and February 2021.

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

Quantitative X-ray element maps of cassiterite crystals from four localities show that Ti, Fe, Nb, Ta, and W define oscillatory zonation patterns and that the cathodoluminescent response is due to a complex interplay between Ti activated emission paired with quenching effects from Fe, Nb, Ta, and W. Sector zonation is commonly highlighted by domains of high Fe, incorporated via a substitution mechanism independent of Nb and Ta. A second form of sector zonation is highlighted by distributions of W separate to the Fe-dominant sector zone. Both sector zones show quenched cathodoluminescence and are indistinguishable under routine SEM CL imaging. For cassiterite already high in Fe (and Nb or Ta), such as in pegmatitic or granitic samples, the internal structure of the grain may remain obscured when imaged by cathodoluminescence techniques, regardless of the presence of sector zonation. Careful petrogenetic assessments using a combination of panchromatic and hyperspectral CL, aided by quantitative elemental X-ray mapping, is a prerequisite step to elucidate cassiterite petrogenetic history and properly characterize these grains for in situ microanalysis. The absence of a clear petrogenetic framework may lead to unknowingly poor spot selection during in situ analyses for geochronology and trace element geochemistry, and/or erroneous interpretations of U-Pb and O isotopic data.

Hospitalized patients are at risk of developing serious multidrug resistant bacterial infections. This risk is heightened in patients who are on mechanical ventilation, are immunocompromised, and/or have chronic comorbidities. We report the case of a 52-year-old critically ill patient with a multidrug resistant Acinetobacter baumannii (MDR-A) respiratory infection who was successfully treated with antibiotics and intravenous and nebulized bacteriophage therapy.

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.

Introduction Skin and soft-tissue infections (SSTIs) are an important cause of infectious disease morbidity among military populations. Due to the high direct and indirect costs associated with SSTIs, particularly with methicillin-resistant Staphylococcus aureus (MRSA) infections, there remains a critical need for the development and evaluation of SSTI prevention strategies among high-risk military personnel. Herein, we review efforts of the Infectious Disease Clinical Research Program (IDCRP) related to the prevention of SSTIs in the military. Methods The IDCRP of the Uniformed Services University has conducted clinical research protocols on SSTI epidemiology and prevention among military personnel since 2009. Observational studies have examined the epidemiology of Staphylococcus aureus colonization and SSTI in training and deployment settings. Two randomized controlled trials of personal hygiene strategies for SSTI prevention at Marine Corps Base Quantico (Virginia) and Fort Benning (Georgia) were performed. Lastly, two vaccine trials have been conducted by the IDCRP, including a Phase 2 S. aureus vaccine trial (currently ongoing) among military trainees. Results Military recruits and deployed personnel experience an intense and prolonged exposure to S. aureus, the major causative agent of SSTI. The burden of S. aureus colonization and SSTI is particularly high in military trainees. Hygiene-based trials for S. aureus decolonization among military trainees were not effective in reducing rates of SSTI. In January 2018, the IDCRP initiated a Phase 2 S. aureus vaccine trial among the US Army Infantry training population at Fort Benning. Conclusions In the military, a disproportionate burden of SSTIs is borne by the recruit population. Strategies relying upon routine application of agents for S. aureus decolonization have not been effective in preventing SSTIs. A novel S. aureus vaccine candidate is being currently evaluated in a military training population and may represent a new opportunity to prevent SSTIs for the military.

BackgroundPlasmodium vivax malaria requires a 2-week course of primaquine (PQ) for radical cure. Evidence suggests that the hepatic isoenzyme cytochrome P450 2D6 (CYP2D6) is the key enzyme required to convert PQ into its active metabolite.MethodsCYP2D6 genotypes and phenotypes of 550 service personnel were determined, and the pharmacokinetics (PK) of a 30-mg oral dose of PQ was measured in 45 volunteers. Blood and urine samples were collected, with PQ and metabolites were measured using ultraperformance liquid chromatography with mass spectrometry.ResultsSeventy-six CYP2D6 genotypes were characterized for 530 service personnel. Of the 515 personnel for whom a single phenotype was predicted, 58% had a normal metabolizer (NM) phenotype, 35% had an intermediate metabolizer (IM) phenotype, 5% had a poor metabolizer (PM) phenotype, and 2% had an ultrametabolizer phenotype. The median PQ area under the concentration time curve from 0 to ∞ was lower for the NM phenotype as compared to the IM or PM phenotypes. The novel 5,6-ortho-quinone was detected in urine but not plasma from all personnel with the NM phenotype.ConclusionThe plasma PK profile suggests PQ metabolism is decreased in personnel with the IM or PM phenotypes as compared to those with the NM phenotype. The finding of 5,6-ortho-quinone, the stable surrogate for the unstable 5-hydroxyprimaquine metabolite, almost exclusively in personnel with the NM phenotype, compared with sporadic or no production in those with the IM or PM phenotypes, provides further evidence for the role of CYP2D6 in radical cure.Clinical Trials RegistrationNCT02960568.

Individuals with methicillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infection (SSTI) can be simultaneously colonized with MRSA on multiple body sites. Using whole genome sequencing (WGS), the intrahost relatedness of MRSA colonization and infection isolates was investigated. In the context of a prospective case–control study of SSTI, we analyzed colonization and infection isolates from US Army Infantry trainees with purulent infection due to MRSA. At the time of clinical presentation for SSTI, culture swabs were obtained from the infection site, as well as from the patient’s nasal, oral, inguinal, and perianal regions. S. aureus culture and susceptibility was performed by standard methods. DNA from MRSA isolates was extracted and libraries were produced. Sequences were generated on an Illumina MiSeq, sequence reads were assembled, and single nucleotide variant (SNV) data were analyzed. Of 74 trainees with MRSA SSTI, 19 (25.7%) were colonized with MRSA. Ten (52.6%) were colonized on more than one body site. Colonization frequency by anatomic site was as follows: inguinal region (33%), nasal region (30%), perianal region (22%), and oral region (14%). A total of 36 MRSA colonization isolates were characterized. The intrahost median number of SNVs between infection and colonization isolates was 17. Among trainees with recurrent MRSA SSTI, limited intrahost diversity suggests that persistent colonization is a major contributor to recurrence risk. Among military trainees with MRSA SSTI, genomic characterization of infection and colonization isolates revealed a high degree of strain relatedness. Single acquisition events may account for MRSA colonization and infection in this population.

A protracted outbreak of New Delhi metallo-beta-lactamase (NDM)-producing carbapenem-resistant Klebsiella pneumoniae, started in Tuscany, Italy, in November 2018 and continued in 2020 and through 2021. To understand the regional emergence and transmission dynamics over time, we collected and sequenced the genomes of 117 extensively drug-resistant, NDM-producing K. pneumoniae isolates cultured over a 20-month period from 76 patients at several health care facilities in South-East Tuscany. All isolates belonged to high-risk clone ST-147 and were typically non-susceptible to all first line antibiotics. Albeit sporadic, resistances to colistin, tigecycline and fosfomycin were also observed as a result of repeated, independent mutations. Genomic analysis revealed that ST-147 isolates circulating in Tuscany were monophyletic, highly genetically related (including a network of 42 patients from the same hospital and sharing nearly identical isolates) and shared a recent ancestor with clinical isolates from the Middle East. While the blaNDM–1 gene was carried by an IncFIB-type plasmid, our investigations revealed that the ST-147 lineage from Italy also acquired a hybrid IncH-type plasmid carrying the 16S methyltransferase armA gene as well as key virulence biomarkers often found in hypervirulent isolates. This plasmid shared extensive homologies with mosaic plasmids circulating globally including from ST-11 and ST-307 convergent lineages. Phenotypically, the carriage of this hybrid plasmid resulted in increased siderophore production but did not confer virulence to the level of an archetypical, hypervirulent K. pneumoniae in a subcutaneous model of infection with immunocompetent CD1 mice. Our findings highlight the importance of performing genomic surveillance to identify emerging threats.Significance StatementCarbapenem-resistant Klebsiella pneumoniae belong to the “critical priority” tier of bacterial pathogens as identified by the World Health Organization. Emerging “high-risk” lineages are responsible for difficult-to-treat, hospital-acquired infections and outbreaks around the globe. By integrating genomic and epidemiological data for isolates collected over 20 months, this study revealed both the high, regional prevalence and the rapid spread, within a single hospital, of K. pneumoniae ST-147 in Italy. Besides resistance to nearly all antibiotics, we showed that this lineage carried a hybrid plasmid harboring a set of biomarker genes previously linked to hypervirulence. Convergence of multidrug resistance and hypervirulence is a major concern and these findings highlight the need for robust, global surveillance to monitor the emergence of high-risk K. pneumoniae.

KPC-82 is a KPC-2 variant identified in a carbapenem-nonsusceptible Citrobacter koseri that confers high-level resistance to ceftazidime-avibactam. Genomic analysis revealed that bla KPC-82 is carried by a chromosomally integrated Tn 4401 transposon (disrupting porin gene phoE ) and evolved by a 6-nucleotide tandem repeat duplication causing a two-amino-acid insertion (Ser-Asp) within the Ala 267 -Ser 275 loop.

Pseudomonas aeruginosa is a leading cause of community-acquired and hospital-acquired infections. Successful treatment is hampered by its remarkable ability to rapidly develop resistance to antimicrobial agents, mostly through mutation. In response, the World Health Organization 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 endeavor.Whole genome sequencing was performed on 3,785 P. aeruginosa housed in our repository. Isolates were cultured from clinical samples collected from healthcare facilities around the world between 2003 and 2017. Core-genome multi-locus sequence typing and high-resolution SNP-based phylogenetic analyses were used to select a panel of 100 strains that captured the genetic diversity of this collection. Comprehensive antibiotic susceptibility testing was also performed using 14 clinically relevant antibiotics.This 100-strain diversity panel contained representative strains from 91 different sequence-types, including genetically distinct isolates from major epidemic clones ST-111, ST-235, ST-244, and ST-253. Seventy-one distinct antibiotic susceptibility profiles were identified ranging from pan-sensitive to pan-resistant. Known resistance alleles as well as the most prevalent mutations underlying the antibiotic susceptibilities were characterized for all isolates.This panel provides a diverse and comprehensive set of P. aeruginosa strains for use in developing solutions to antibiotic resistance. The isolates, and all available meta-data including genome sequences, are available to industry, academic institutions, federal and other laboratories at no additional cost.ImportancePseudomonas aeruginosa is one of the most important human pathogens and a leading target in the development of new drugs and therapeutics. This species displays a remarkable level of diversity and any potential therapeutic must contend with this characteristic to ensure it retains efficacy across different strains. To date, only limited panels of P. aeruginosa are available for testing, and none have been designed to capture the genetic diversity of the species. The panel described herein has been designed to address this shortcoming by providing a set of 100 distinct strains that greatly captures the diversity of the species. This panel will be of significant value to research groups working on this important pathogen, both for research purposes and in the development of new diagnostics and countermeasures.

Ongoing surveillance detected a SARS-CoV-2 B.1.1.220 variant carrying the E484K substitution in four patients from a hospital network in upstate New York. Patients reported no travel history and shared no obvious epidemiological linkage. A search of online databases identified 12 additional B.1.1.220 with E484K, all of which were detected in New York since December 2020. Detailed genomic analyses suggests that the mutation has emerged independently in at least two different B.1.1.220 strains in this region.

The purpose of this study was to evaluate the hypothesis that a novel high-repetition, low-resistance back squat training protocol, designed to stimulate high-intensity interval training, improves 5-km run performance. Fifteen runners [4 male, 11 female; 150 + minutes of endurance exercise/week; age = 22.7 ± 2.0 y; 21.5 ± 2.2 kg/m(2) BMI] in this single-group test-retest design completed two weeks of back squats consisting of three sets of 15–24 repetitions at 60% of estimated one-repetition max (1RM), three times per week (1–2 days of rest between sessions). Outcome tests included a 5-km outdoor timed run, laboratory indirect calorimetry to quantify substrate oxidation rates during steady-state submaximal exercise (60% and 70% heart rate max (HRmax)), and estimated 1RM for back squats. Back squat estimated 1RM increased by 20% (58.3 ± 18.5 to 70.2 ± 16.7 kg, P < 0.001). However, 5-km run times due to the back squat protocol did not significantly change (Pre-Squats: 23.9 ± 5.0 vs. Post-Squats: 23.7 ± 4.3 minutes, P = 0.71). Likewise, the squat training program did not significantly alter carbohydrate or lipid oxidation rates during steady-state submaximal exercise at 60% or 70% of HRmax (P values ranged from 0.36 – 0.99). Short term high-repetition back squat training does not appear to impact 5-km run performance or substrate utilization during submaximal exercise.

Significance Carbapenem-resistant Klebsiella pneumoniae belongs to the “critical-priority” tier of bacterial pathogens as identified by the World Health Organization. Emerging “high-risk” lineages are responsible for difficult-to-treat, hospital-acquired infections and outbreaks around the globe. By integrating genomic and epidemiological data for isolates collected over 20 mo, this study revealed both the high, regional prevalence and the rapid spread, within a single hospital, of K. pneumoniae ST-147 in Italy. Besides resistance to nearly all antibiotics, this lineage carried a hybrid plasmid harboring a set of biomarker genes previously linked to hypervirulence. Convergence of resistance and virulence determinants is a major concern and these findings highlight the need for robust, global surveillance to monitor the emergence of high-risk K. pneumoniae., A protracted outbreak of New Delhi metallo-β-lactamase (NDM)–producing carbapenem-resistant Klebsiella pneumoniae started in Tuscany, Italy, in November 2018 and continued in 2020 and through 2021. To understand the regional emergence and transmission dynamics over time, we collected and sequenced the genomes of 117 extensively drug-resistant, NDM-producing K. pneumoniae isolates cultured over a 20-mo period from 76 patients at several healthcare facilities in southeast Tuscany. All isolates belonged to high-risk clone ST-147 and were typically nonsusceptible to all first-line antibiotics. Albeit sporadic, resistances to colistin, tigecycline, and fosfomycin were also observed as a result of repeated, independent mutations. Genomic analysis revealed that ST-147 isolates circulating in Tuscany were monophyletic and highly genetically related (including a network of 42 patients from the same hospital and sharing nearly identical isolates), and shared a recent ancestor with clinical isolates from the Middle East. While the blaNDM-1 gene was carried by an IncFIB-type plasmid, our investigations revealed that the ST-147 lineage from Italy also acquired a hybrid IncFIB/IncHIB–type plasmid carrying the 16S methyltransferase armA gene as well as key virulence biomarkers often found in hypervirulent isolates. This plasmid shared extensive homologies with mosaic plasmids circulating globally including from ST-11 and ST-307 convergent lineages. Phenotypically, the carriage of this hybrid plasmid resulted in increased siderophore production but did not confer virulence to the level of an archetypical, hypervirulent K. pneumoniae in a subcutaneous model of infection with immunocompetent CD1 mice. Our findings highlight the importance of performing genomic surveillance to identify emerging threats.

Background Military trainees are at increased risk for Staphylococcus aureus colonization and infection. Disease prevention strategies are needed, but a S. aureus vaccine does not currently exist. Methods We enrolled US Army Infantry trainees (Fort Benning, GA) in a phase 2, randomized, double-blind, placebo-controlled trial of NDV-3A, a vaccine containing a recombinant adhesin/invasion protein of Candida albicans that has structural similarity to the S. aureus protein clumping factor A. Study participants received one intramuscular dose of NDV-3A or placebo (adjuvant alone) within 72 h of arrival on base. Longitudinal nasal and oral (throat) swabs were collected throughout the 14-week Infantry training cycle. Safety, immunogenicity, and efficacy of NDV-3A against S. aureus nasal / oral acquisition were the endpoints. Results The NDV-3A candidate had minimal reactogenicity and elicited robust antigen-specific B- and T-cell responses. During the 56-day post-vaccination period, there was no difference in the incidence of S. aureus nasal acquisition between those who were randomized to receive NDV-3A vs. placebo (25.6% vs. 29.1%; vaccine efficacy [VE]: 12.1%; p = 0.31). In time-to-event analysis, there was no difference between study groups with respect to the S. aureus colonization-free interval (VE: 13%; p = 0.29). Similarly, the efficacy of NDV-3A against S. aureus oral acquisition was poor (VE: 2.4%; p = 0.52). Conclusions A single dose of NDV-3A did not prevent nasal nor oral acquisition of S. aureus in a population of military trainees at high risk for colonization.

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.

Decision-making related to the utilization of host-nation medical resources in austere forward-deployed environments is complex. Clinical circumstances, local medical intelligence availability, transportation assets, uncertainty regarding standard-of-care variations, military/host-nation funding complications, and regional security concerns all factor into consideration. A case of a U.S. active duty military service member who suffered a cardiac arrest on a military base in Southwest Asia is described in this report. After return of circulation following defibrillation, he was administered thrombolytic therapy for an electrocardiogram-identified ST-elevation myocardial infarction and transported to a local host-nation cardiac hospital for emergent percutaneous coronary intervention. During his subsequent transportation back to the USA, surveillance testing identified that he was colonized with a rare strain of Pseudomonas aeruginosa, demonstrating New Delhi metallo-beta-lactamase-1 and 16S RNA methyltransferase-2 enzymes, which confer significant resistance to carbapenem and aminoglycoside antibiotics, respectively.1–3 This combination of antibiotic resistance has been reported very rarely in the medical literature and has never been reported within the deployed military health system until now. The risk of exposure to multidrug-resistant organisms was not a factor initially considered in the decision to utilize host-nation medical resources in this case, which provided lesson learned and raised new questions, for future operational medical planning.

Background Military trainees are at increased risk for methicillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infection (SSTI). Whole genome sequencing (WGS) can refine our understanding of MRSA transmission and microevolution in congregate settings. Methods We conducted a prospective case-control study of SSTI among US Army infantry trainees at Fort Benning, Georgia, from July 2012 to December 2014. We identified clusters of USA300 MRSA SSTI within select training classes and performed WGS on clinical isolates. We then linked genomic, phylogenetic, epidemiologic, and clinical data in order to evaluate intra- and interclass disease transmission. Furthermore, among cases of recurrent MRSA SSTI, we evaluated the intrahost relatedness of infecting strains. Results Nine training classes with ≥5 cases of USA300 MRSA SSTI were selected. Eighty USA300 MRSA clinical isolates from 74 trainees, 6 (8.1%) of whom had recurrent infection, were subjected to WGS. We identified 2719 single nucleotide variants (SNVs). The overall median (range) SNV difference between isolates was 173 (1-339). Intraclass median SNV differences ranged from 23 to 245. Two phylogenetic clusters were suggestive of interclass MRSA transmission. One of these clusters stemmed from 2 classes that were separated by a 13-month period but housed in the same barracks. Among trainees with recurrent MRSA SSTI, the intrahost median SNV difference was 7.5 (1-48). Conclusions Application of WGS revealed intra- and interclass transmission of MRSA among military trainees. An interclass cluster between 2 noncontemporaneous classes suggests a long-term reservoir for MRSA in this setting.

The worst Ebola virus disease (EVD) outbreak in history has resulted in more than 28,000 cases and 11,000 deaths. We present the final results of two phase 1 trials of an attenuated, replication-competent, recombinant vesicular stomatitis virus (rVSV)-based vaccine candidate designed to prevent EVD.We conducted two phase 1, placebo-controlled, double-blind, dose-escalation trials of an rVSV-based vaccine candidate expressing the glycoprotein of a Zaire strain of Ebola virus (ZEBOV). A total of 39 adults at each site (78 participants in all) were consecutively enrolled into groups of 13. At each site, volunteers received one of three doses of the rVSV-ZEBOV vaccine (3 million plaque-forming units [PFU], 20 million PFU, or 100 million PFU) or placebo. Volunteers at one of the sites received a second dose at day 28. Safety and immunogenicity were assessed.The most common adverse events were injection-site pain, fatigue, myalgia, and headache. Transient rVSV viremia was noted in all the vaccine recipients after dose 1. The rates of adverse events and viremia were lower after the second dose than after the first dose. By day 28, all the vaccine recipients had seroconversion as assessed by an enzyme-linked immunosorbent assay (ELISA) against the glycoprotein of the ZEBOV-Kikwit strain. At day 28, geometric mean titers of antibodies against ZEBOV glycoprotein were higher in the groups that received 20 million PFU or 100 million PFU than in the group that received 3 million PFU, as assessed by ELISA and by pseudovirion neutralization assay. A second dose at 28 days after dose 1 significantly increased antibody titers at day 56, but the effect was diminished at 6 months.This Ebola vaccine candidate elicited anti-Ebola antibody responses. After vaccination, rVSV viremia occurred frequently but was transient. These results support further evaluation of the vaccine dose of 20 million PFU for preexposure prophylaxis and suggest that a second dose may boost antibody responses. (Funded by the National Institutes of Health and others; rVSV∆G-ZEBOV-GP ClinicalTrials.gov numbers, NCT02269423 and NCT02280408 .).

BackgroundMRSA skin and soft tissue infection (SSTI) is a significant cause of morbidity in military trainees. To guide interventions, it is critical we understand the epidemiology of MRSA in this population.MethodsTwo cohorts (‘companies’) of US Army Infantry trainees (N=343) at Fort Benning, GA, USA, were followed during their training cycles (Jun.-Dec. 2015). Trainees had nares, oropharynx, perianal and inguinal areas swabbed for MRSA colonization at five ∼2-4 week intervals, and monitored for SSTI throughout training. Epidemiological data were collected. Isolates were sequenced using Illumina HiSeq and NovaSeq. Single-nucleotide polymorphisms and clusters were identified. Multi-locus sequence type (MLST) and antimicrobial resistance genes were predicted from de novo assemblies.Results87 trainees were positive at least once for MRSA (12 had SSTI, 2 without any colonization). Excluding those positive at baseline, 43.7% were colonized within the first month of training. 244/254 samples were successfully sequenced (including all SSTI). ST8 (n=135, 100% of SSTI), ST5 (n=81) and ST87 (n=21) were the most represented. Three main Clusters were identified, largely corresponding to these STs. Sub-analyses within Clusters showed multiple importations of MRSA, with transmission subsequently predominantly within, rather than between, platoons in each company. Over 50% of trainees were colonized only at other anatomical sites; restricting analyses to nares missed substantial transmission.ConclusionsSerial importations of MRSA into this high-risk setting likely contribute to the ongoing burden of MRSA colonization and infection among military trainees. Sampling multiple anatomical sites is critical for comprehensive characterization of MRSA transmissionSummaryUS Infantry trainees were followed through training for MRSA skin and soft tissue infection, swabbing for colonization at 2-4 week intervals. Sequencing suggests serial importations of diverse strains on base, followed by transmission mostly within platoons, involving multiple anatomical sites.

Selective field ionization is used to determine the state or distribution of states to which a Rydberg atom is excited. By evolving a small perturbation to the ramped electric field using a genetic algorithm, the shape of the time-resolved ionization signal can be controlled. This allows for separation of signals from pairs of states that would be indistinguishable with unperturbed selective field ionization. Measurements and calculations are presented that demonstrate this technique and shed light on how the perturbation directs the pathway of the electron to ionization. Pseudocode for the genetic algorithm is provided. Using the improved resolution afforded by this technique, quantitative measurements of the $36p_{3/2}+36p_{3/2}\rightarrow 36s_{1/2}+37s_{1/2}$ dipole-dipole interaction are made.

Bacteria often exist in polymicrobial communities where they compete for limited resources. Intrinsic to this competition is the ability of some species to inhibit or kill their competitors. This phenomenon is pervasive throughout the human body where commensal bacteria block the colonization of incoming microorganisms. In this regard, molecular epidemiological and microbiota-based studies suggest that species-specific interactions play a critical role in the prevention of nasal colonization of the opportunistic pathogen Staphylococcus aureus. Despite this, S. aureus exists as part of the microbiota of ∼25% of the population, suggesting that the interplay between S. aureus and commensals can be complex. Microbiota studies indicate that several bacterial genera are negatively correlated with S. aureus colonization. While these studies paint a broad overview of bacterial presence, they often fail to identify individual species-specific interactions; a greater insight in this area could aid the development of novel antimicrobials. As a proof of concept study designed to identify individual bacterial species that possess anti-S. aureus activity, we screened a small collection of clinical isolates from the Walter Reed National Military Medical Center for the ability to inhibit multiple S. aureus strains. We found that the majority of the isolates (82%) inhibited at least one S. aureus strain; 23% inhibited all S. aureus strains tested. In total, seven isolates mediated inhibitory activity that was independent of physical contact with S. aureus, and seven isolates mediated bactericidal activity. 16S rRNA based-sequencing revealed that the inhibitory isolates belonged to the Acinetobacter, Agromyces, Corynebacterium, Microbacteria, Mycobacterium, and Staphylococcus genera. Unexpectedly, these included seven distinct Acinetobacter baumannii isolates, all of which showed heterogeneous degrees of anti-S. aureus activity. Defined mechanistic studies on specific isolates revealed that the inhibitory activity was retained in conditioned cell free medium (CCFM) derived from the isolates. Furthermore, CCFM obtained from S. saprophyticus significantly decreased mortality of S. aureus-infected Galleria mellonella caterpillars. While future studies will seek to define the molecular mechanisms of the inhibitory activities, our current findings support the study of polymicrobial interactions as a strategy to understand bacterial competition and to identify novel therapeutics against S. aureus and other pathogens.

Seroepidemiological studies on the prevalence of antibodies to malaria antigens are primarily conducted on individuals from regions of endemicity. It is therefore difficult to accurately correlate the antibody responses to the timing and number of prior malaria infections. This study was undertaken to assess the evolution of antibodies to the dominant surface antigens of Plasmodium vivax and P. falciparum following controlled human malaria infection (CHMI) in malaria-naive individuals. Serum samples from malaria-naive adults, collected before and after CHMI with either P. vivax (n = 18) or P. falciparum (n = 18), were tested for the presence of antibodies to the circumsporozoite protein (CSP) and the 42-kDa fragment of merozoite surface protein 1 (MSP-142) of P. vivax and P. falciparum using an enzyme-linked immunosorbent assay (ELISA). Approximately 1 month following CHMI with either P. vivax or P. falciparum, >60% of subjects seroconverted to homologous CSP and MSP-1. More than 50% of the subjects demonstrated reactivity to heterologous CSP and MSP-142, and a similar proportion of subjects remained seropositive to homologous MSP-142 >5 months after CHMI. Computational analysis provides insight into the presence of cross-reactive responses. The presence of long-lived and heterologous reactivity and its functional significance, if any, need to be taken into account while evaluating malaria exposure in field settings.

Systematic density functional theory calculations that treat the strong on-site 3d electron–electron interactions on iron via a Hubbard Ueff = 3.0 eV and the van der Waals (vdW) interactions between the substrate and adsorbate within the vdW-DF framework are employed to study the adsorption of the iron porphyrin (FeP) molecule to the Pt(111) surface. The more accurate vdW-DF-optPBE and vdW-DF-optB88 functionals found the same binding site to be the most stable and yielded binding energies that were within ∼20% of each other, whereas the binding energies computed with the vdW-DF-revPBE functional were substantially weaker. This work highlights the importance of vdW interactions for organometallic molecules chemisorbed to transition metal surfaces. The stability of the binding sites was found to depend upon the number of Fe–Pt and C–Pt bonds that were formed. Whereas in the gas phase the most stable spin state of FeP is the intermediate spin S = 1 state, the high spin S = 2 state is preferred for the FeP–Pt...

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.

Graphene oxide electrochemically reduced from aqueous and nonaqueous graphene oxide suspensions was compared. The protocol that utilized the nonaqueous suspension resulted in a more reproducibly complete covering of the underlying Pt electrode while still retaining its electrochemical integrity. The hydrogen evolution reaction was found to be detrimental to achieving complete electrode coverage from the aqueous suspension due to H 2 bubbles physically dislodging previously reduced graphene oxide. However, since this parasitic reaction does not proceed as readily in organic solvents such as DMF, the nonaqueous reduction was able to form a complete layer once the aqueous suspension was drop-casted and dried on the Pt electrode. The graphene oxide-coated electrodes resulting from both aqueous and nonaqueous protocols were compared using electrochemistry, SEM, AFM, and XPS. Overall, the sample produced from the nonaqueous suspension possessed a significantly rougher morphology and was determined to possess a higher amount of reduced carbon than that produced using the aqueous suspension.