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Objectives To determine the susceptibility profiles and the resistome of Pseudomonas aeruginosa isolates from European ICUs during a prospective cohort study (ASPIRE-ICU). Methods 723 isolates from respiratory samples or perianal swabs of 402 patients from 29 sites in 11 countries were studied. MICs of 12 antibiotics were determined by broth microdilution. Horizontally acquired β-lactamases were analysed through phenotypic and genetic assays. The first respiratory isolates from 105 patients providing such samples were analysed through WGS, including the analysis of the resistome and a previously defined genotypic resistance score. Spontaneous mutant frequencies and the genetic basis of hypermutation were assessed. Results All agents except colistin showed resistance rates above 20%, including ceftolozane/tazobactam and ceftazidime/avibactam. 24.9% of the isolates were XDR, with a wide intercountry variation (0%–62.5%). 13.2% of the isolates were classified as DTR (difficult-to-treat resistance). 21.4% of the isolates produced ESBLs (mostly PER-1) or carbapenemases (mostly NDM-1, VIM-1/2 and GES-5). WGS showed that these determinants were linked to high-risk clones (particularly ST235 and ST654). WGS revealed a wide repertoire of mutation-driven resistance mechanisms, with multiple lineage-specific mutations. The most frequently mutated genes were gyrA, parC, oprD, mexZ, nalD and parS, but only two of the isolates were hypermutable. Finally, a good accuracy of the genotypic score to predict susceptibility (91%–100%) and resistance (94%–100%) was documented. Conclusions An overall high prevalence of resistance is documented European ICUs, but with a wide intercountry variability determined by the dissemination of XDR high-risk clones, arguing for the need to reinforce infection control measures.

We investigated the performance of the Xpert methicillin-resistant Staphylococcus aureus (MRSA)/S. aureus skin and soft tissue (SSTI) quantitative PCR (qPCR) assay in SAATELLITE, a multicenter, double-blind, phase 2 study of suvratoxumab, a monoclonal antibody (MAb) targeting S. aureus alpha-toxin, for reducing the incidence of S. aureus pneumonia. The assay was used to detect methicillin-susceptible S. aureus (MSSA) and MRSA in lower respiratory tract (LRT) samples from mechanically ventilated patients. LRT culture results were compared with S. aureus protein A (spa) gene cycle threshold (C-T) values. Receiver operating characteristic (ROC) and Youden index were used to determine the C-T cutoff for best separation of culture-S. aureus-negative and S. aureus-positive patients. Of 720 screened subjects, 299 (415%) were S. aureus positive by qPCR, of whom 209 had culture data: 162 (77.5%) were S. aureus positive and 47 (22.5%) were S. aureus negative. Culture results were negatively affected by antibiotic use and cross-laboratory variability. An inverse linear correlation was observed between C-T values and quantitative S. aureus culture results. A spa Cr value of 29 (approximate to 2 x 10(3) CFU/mL) served as the best cutoff for separation between culture-negative and culture-positive samples. The associated area under the ROC curve was 83.8% (95% confidence interval (CI), 78 to 90%). Suvratoxumab provided greater reduction in S. aureus pneumonia or death than placebo in subjects with low S. aureus load (C-T >= 29; relative risk reduction [RRR], 50.0%; 90% CI, 2.7 to 74.4%) versus the total study population (RRR, 252%; 90% CI, -43 to 46.4%). The qPCR assay was easy to perform, sensitive, and standardized and provided better sensitivity than conventional culture for S. aureus detection. Quantitative PCR C-T output correlated with suvratoxumab efficacy in reducing S. aureus pneumonia incidence or death in S. aureus-colonized, mechanically ventilated patients.

Background Respiratory syncytial virus (RSV) is a global cause of severe respiratory morbidity and mortality in infants. While preventive and therapeutic interventions are being developed, including antivirals, vaccines and monoclonal antibodies, little is known about the global molecular epidemiology of RSV. INFORM is a prospective, multicenter, global clinical study performed by ReSViNET to investigate the worldwide molecular diversity of RSV isolates collected from children less than 5 years of age. Methods The INFORM study is performed in 17 countries spanning all inhabited continents and will provide insight into the molecular epidemiology of circulating RSV strains worldwide. Sequencing of > 4000 RSV-positive respiratory samples is planned to detect temporal and geographical molecular patterns on a molecular level over five consecutive years. Additionally, RSV will be cultured from a subset of samples to study the functional implications of specific mutations in the viral genome including viral fitness and susceptibility to different monoclonal antibodies. Discussion The sequencing and functional results will be used to investigate susceptibility and resistance to novel RSV preventive or therapeutic interventions. Finally, a repository of globally collected RSV strains and a database of RSV sequences will be created.

The lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multi-drug-resistant Gram-negative bacteria. Here, we report two novel small-molecule inhibitors (compounds 1 and 2) of the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically that the antibacterial activities of the compounds against efflux-deficient Escherichia coli are mediated by LpxA inhibition. Consistently, the compounds inhibited the LpxA enzymatic reaction in vitro. Intriguingly, using biochemical, biophysical, and structural characterization, we reveal two distinct mechanisms of LpxA inhibition; compound 1 is a substrate-competitive inhibitor targeting apo LpxA, and compound 2 is an uncompetitive inhibitor targeting the LpxA/product complex. Compound 2 exhibited more favorable biological and physicochemical properties than compound 1 and was optimized using structural information to achieve improved antibacterial activity against wild-type E. coli. These results show that LpxA is a promising antibacterial target and imply the advantages of targeting enzyme/product complexes in drug discovery.

Robust, quantitative serology assays are required to accurately measure antibody levels following vaccination and natural infection. We present validation of a quantitative, multiplex, SARS-CoV-2, electrochemiluminescent (ECL) serology assay; show correlation with two established SARS-CoV-2 immunoassays; and present calibration results for two SARS-CoV-2 reference standards.Precision, dilutional linearity, ruggedness, analytical sensitivity and specificity were evaluated. Clinical sensitivity and specificity were assessed using serum from prepandemic and SARS-CoV-2 polymerase chain reaction (PCR)-positive patient samples. Assay concordance to the established Roche Elecsys® Anti-SARS-CoV-2 immunoassay and a live-virus microneutralisation (MN) assay was evaluated.Standard curves demonstrated the assay can quantify SARS-CoV-2 antibody levels over a broad range. Assay precision (10.2-15.1% variability), dilutional linearity (≤ 1.16-fold bias per 10-fold increase in dilution), ruggedness (0.89-1.18 overall fold difference), relative accuracy (107-118%) and robust selectivity (102-104%) were demonstrated. Analytical sensitivity was 7, 13 and 7 arbitrary units mLThe multiplex SARS-CoV-2 ECL serology assay is suitable for efficient, reproducible measurement of antibodies to SARS-CoV-2 antigens in human sera, supporting its use in clinical trials and sero-epidemiology studies.

Summary Background Staphylococcus aureus remains a common cause of ventilator-associated pneumonia, with little change in incidence over the past 15 years. We aimed to evaluate the efficacy of suvratoxumab, a monoclonal antibody targeting the α toxin, in reducing the incidence of S aureus pneumonia in patients in the intensive care unit (ICU) who are on mechanical ventilation. Methods We did a multicentre, randomised, double-blind, placebo-controlled, parallel-group, phase 2 pilot trial at 31 hospitals in Belgium, the Czech Republic, France, Germany, Greece, Hungary, Portugal, Spain, and Switzerland. Eligible patients were in the ICU, aged ≥18 years, were intubated and on mechanical ventilation, were positive for S aureus colonisation of the lower respiratory tract, as assessed by quantitative PCR (qPCR) analysis of endotracheal aspirate, and had not been diagnosed with new-onset pneumonia. Patients were excluded if they had confirmed or suspected acute ongoing staphylococcal disease; had received antibiotics for S aureus infection for more than 48 h within 72 h of randomisation; had a Clinical Pulmonary Infection Score of 6 or higher; had an acute physiology and chronic health evaluation II score of 25 or higher with a Glasgow coma scale (GCS) score of more than 5, or an acute physiology and chronic health evaluation II score of at least 30 with a GCS score of 5 or less; had a Sequential Organ Failure Assessment score of 9 or higher; or had active pulmonary disease that would impair the ability to diagnose pneumonia. Colonised patients were randomly assigned (1:1:1), by use of an interactive voice or web response system, to receive either a single intravenous infusion of suvratoxumab 2000 mg, suvratoxumab 5000 mg, or placebo. Randomisation was done in blocks of size four, stratified by country and by whether patients had received systemic antibiotics for S aureus infection. Patients, investigators, and study staff involved in the treatment or clinical evaluation of patients were masked to patient assignment. The primary efficacy endpoint was the incidence of S aureus pneumonia at 30 days, as determined by a masked independent endpoint adjudication committee, in all patients who received their assigned treatment (modified intention-to-treat [ITT] population). Primary safety endpoints were the incidence of treatment-emergent adverse events at 30 days, 90 days, and 190 days after treatment, and the incidence of treatment-emergent serious adverse events, adverse events of special interest, and new-onset chronic disease at 190 days after treatment. All primary safety endpoints were assessed in the modified ITT population. This trial is registered with ClinicalTrials.gov ( NCT02296320 ) and the EudraCT database (2014-001097-34). Findings Between Oct 10, 2014, and April 1, 2018, 767 patients were screened, of whom 213 patients with confirmed S aureus colonisation of the lower respiratory tract were randomly assigned to the suvratoxumab 2000 mg group (n=15), the suvratoxumab 5000 mg group (n=96), or the placebo group (n=102). Two patients in the placebo group did not receive treatment after randomisation because their clinical conditions changed and they no longer met the eligibility criteria for dosing. As adjudicated by the data monitoring committee at an interim analysis, the suvratoxumab 2000 mg group was discontinued on the basis of predefined pharmacokinetic criteria. At 30 days after treatment, 17 (18%) of 96 patients in the suvratoxumab 5000 mg group and 26 (26%) of 100 patients in the placebo group had developed S aureus pneumonia (relative risk reduction 31·9% [90% CI −7·5 to 56·8], p=0·17). The incidence of treatment-emergent adverse events at 30 days were similar between the suvratoxumab 5000 mg group (87 [91%]) and the placebo group (90 [90%]). The incidence of treatment-emergent serious adverse events at 30 days were also similar between the suvratoxumab 5000 mg group (36 [38%]) and the placebo group (32 [32%]). No significant difference in the incidence of treatment-emergent adverse events between the two groups at 90 days (89 [93%] in the suvratoxumab 5000 mg group vs 92 [92%] in the placebo group) and at 190 days (93 [94%] vs 93 [93%]) was observed. 40 (40%) patients in the placebo group and 50 (52%) in the suvratoxumab 5000 mg group had a serious adverse event at 190 days. In the suvratoxumab 5000 mg group, one (1%) patient reported at least one treatment-emergent serious adverse event related to treatment, two (2%) patients reported an adverse event of special interest, and two (2%) reported a new-onset chronic disease. Interpretation In patients in the ICU receiving mechanical ventilation with qPCR-confirmed S aureus colonisation of the lower respiratory tract, the incidence of S aureus pneumonia at 30 days was not significantly lower following treatment with 5000 mg suvratoxumab than with placebo. Despite these negative results, monoclonal antibodies still represent one promising therapeutic option to reduce antibiotic consumption that require further exploration and studies. Funding AstraZeneca, with support from the Innovative Medicines Initiative Joint Undertaking.

IntroductionPseudomonas aeruginosais a common cause of ventilator-associated pneumonia (VAP). Rapid and accurate detection of lower respiratory tract colonization and/or infection withP. aeruginosamay advise targeted preventive (antibody-based) strategies and antibiotic therapy. To investigate this, we compared semi-quantitative culture results from 80 endotracheal aspirates (ETA) collected from mechanically-ventilated patients, to two culture and two non-culture-based methods for detection ofP. aeruginosa.MethodsP. aeruginosa-positive (n = 40) and -negative (n = 40) ETAs from mechanically ventilated patients analyzed initally by (i) routine semi-quantitative culture, were further analyzed with (ii) quantitative culture on chromogenic ChromIDP. aeruginosaand blood agar; (iii) enrichment in brain heart infusion broth followed by plating on blood agar and ChromIDP. aeruginosa; (iv) O-antigen acetylase gene-based TaqMan qPCR; and (v) GeneXpert PA PCR assay.ResultsOf the 80 ETA samples included, one sample that was negative forP. aeruginosaby semi-quantitative culture was found to be positive by the other four methods, and was included in an “extended” gold standard panel. Based on this extended gold standard, both semi-quantitative culture and the GeneXpert PA assay showed 97.6% sensitivity and 100% specificity. The quantitative culture, enrichment culture and O-antigen acetylase gene-based TaqMan qPCR had a sensitivity of 97.6%, 89.5%, 92.7%, and a specificity of 97.4%, 100%, and 71.1%, respectively.ConclusionThis first evaluation of the GeneXpert PA assay with ETA samples found it to be as sensitive and specific as the routine, hospital-based semi-quantitative culture method. Additionally, the GeneXpert PA assay is easy to perform (hands-on time ≈ 5 min) and rapid (≈ 55 min assay time). The combination of the high sensitivity and high specificity together with the rapid acquisition of results makes the GeneXpert PA assay a highly recommended screening technique. Where this equipment is not available, semi-quantitative culture remains the most sensitive of the culture methods evaluated here forP. aeruginosadetection in ETA samples.

It is well established that antibiotic treatment selects for resistance, but the dynamics of this process during infections are poorly understood. Here we map the responses of Pseudomonas aeruginosa to treatment in high definition during a lung infection of a single ICU patient. Host immunity and antibiotic therapy with meropenem suppressed P. aeruginosa, but a second wave of infection emerged due to the growth of oprD and wbpM meropenem resistant mutants that evolved in situ. Selection then led to a loss of resistance by decreasing the prevalence of low fitness oprD mutants, increasing the frequency of high fitness mutants lacking the MexAB-OprM efflux pump, and decreasing the copy number of a multidrug resistance plasmid. Ultimately, host immunity suppressed wbpM mutants with high meropenem resistance and fitness. Our study highlights how natural selection and host immunity interact to drive both the rapid rise, and fall, of resistance during infection., Here, following a patient with severe acute Pseudomonas aeruginosa infection, the authors combine comprehensive isolate characterization from lung and gut samples (>100 isolates) and patient clinical data to provide insights into bacterial responses to antibiotic therapy.

Respiratory syncytial virus (RSV) is a significant cause of lower respiratory tract infection in infants and elderly. To understand the evolution of neutralizing epitopes on the RSV glycoprotein (G) and fusion (F) proteins, we conducted a multi-year surveillance program (OUTSMART-RSV) in the US. Analysis of 1,146 RSV samples from 2015–2017 revealed a slight shift in prevalence from RSV A (58.7%) to B (53.7%) between the two seasons. RSV B was more prevalent in elderly (52.9% and 73.4%). Approximately 1% of the samples contained both RSV A and B viruses. All RSV A isolates were ON1 and almost all the B isolates were BA9 genotypes. Compared with the 2013 reference sequences, changes at the F antigenic sites of RSV B were greater than RSV A, which mainly occurred at antigenic sites V (L172Q/S173L at 99.6%), Ø (I206M/Q209K at 18.6%) and IV (E463D at 7%) of RSV B F. Sequence diversities in the G protein second hypervariable region were observed in the duplicated regions for RSV A and B, and at the G stop codon resulting in extension of 7 amino acids (22.1%) for RSV B in 2016–17. Thus, RSV surface glycoproteins are continuously evolving, and continued surveillance is important for the clinical evaluation of immunoprophylactic products.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection among infants and young children, resulting in annual epidemics worldwide. INFORM-RSV is a multiyear clinical study designed to describe the global molecular epidemiology of RSV in children under 5 years of age by monitoring temporal and geographical evolution of current circulating RSV strains, F protein antigenic sites, and their relationships with clinical features of RSV disease. During the pilot season (2017–2018), 410 RSV G-F gene sequences were obtained from 476 RSV-positive nasal samples collected from 8 countries (United Kingdom, Spain, The Netherlands, Finland, Japan, Brazil, South Africa, and Australia)., Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection among infants and young children, resulting in annual epidemics worldwide. INFORM-RSV is a multiyear clinical study designed to describe the global molecular epidemiology of RSV in children under 5 years of age by monitoring temporal and geographical evolution of current circulating RSV strains, F protein antigenic sites, and their relationships with clinical features of RSV disease. During the pilot season (2017–2018), 410 RSV G-F gene sequences were obtained from 476 RSV-positive nasal samples collected from 8 countries (United Kingdom, Spain, The Netherlands, Finland, Japan, Brazil, South Africa, and Australia). RSV B (all BA9 genotype) predominated over RSV A (all ON1 genotype) globally (69.0% versus 31.0%) and in all countries except South Africa. Geographic clustering patterns highlighted wide transmission and continued evolution with viral spread. Most RSV strains were from infants of 24 h (70.5%), with no differences in subtype distribution. Compared to 2013 reference sequences, variations at F protein antigenic sites were observed for both RSV A and B strains, with high-frequency polymorphisms at antigenic site Ø (I206M/Q209R) and site V (L172Q/S173L/K191R) in RSV B strains. The INFORM-RSV 2017–2018 pilot season establishes an important molecular baseline of RSV strain distribution and sequence variability with which to track the emergence of new strains and provide an early warning system of neutralization escape variants that may impact transmission or the effectiveness of vaccines and MAbs under development.

It is well established that antibiotic treatment selects for resistance in pathogenic bacteria. However, the evolutionary responses of pathogen populations to antibiotic treatment during infections remain poorly resolved, especially in acute infections. Here we map the evolutionary responses to treatment in high definition through genomic and phenotypic characterization of >100 isolates from a patient with P. aeruginosa pneumonia. Antibiotic therapy (meropenem, colistin) caused a rapid crash of the P. aeruginosa population in the lung, but this decline was followed by the spread of meropenem resistance mutations that restrict antibiotic uptake (oprD) or modify LPS biosynthesis (wbpM). Low fitness strains with high-level meropenem resistance (oprD) were then replaced by high fitness strains with ‘anti-resistance’ mutations in the MexAB-OprM efflux pump, causing a rapid decline in resistance to both meropenem and a collateral loss of resistance to a broad spectrum of antibiotics. In contrast, we did not observe any evolutionary responses to antibiotic treatment in the intestinal population of P. aeruginosa. Carbapenem antibiotics are key to the treatment of infections caused by Gram negative pathogens, and our work highlights the ability of natural selection to drive both the rapid rise and fall of carbapenem resistance during acute infections.

Background. The emergence and spread of bacteria with resistance to multiple antimicrobial agents is raising the mortality rates associated with bacterial infections, and poses a fundamental threat to human health. Pseudomonas aeruginosais an opportunistic human pathogen that is responsible for 10-15% of health-care associated infections worldwide, and is known for its ability to rapidly develop enhanced resistance during the course of treating an infection. Methods. P. aeruginosa isolates have been collected from ICU patients in hospitals across Europe over a longitudinal sampling of short-term acute respiratory infection. Twelve isolates per patient per sampling of infection have been acquired. Genome sequencing and high-throughput phenotyping of growth rate and antibiotic resistance profile has been carried out. Results and conclusions. Using high-throughput phenotyping and genome sequence data we have been able to characterise population diversity and variation in phenotypic resistance profile, growth rate and acquired resistance gene content of these isolates. A gradient of diversity was found within patient infections, ranging from monoclonal to multiple strains present. Large within-patient population diversity in phenotypic resistance profile and acquired resistance gene content could be observed in multiple sequence-type infections. This research has been carried out in collaboration with the COMBACTE-MAGNET research consortium (Combatting Bacterial Resistance in Europe – Molecules against Gram-Negative Infections).

The lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multidrug-resistant Gram-negative bacteria. Here, we report two novel small-molecule inhibitors (compounds 1 and 2) of the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically that the antibacterial activities of the compounds against efflux-deficient Escherichia coli are mediated by LpxA inhibition. Consistently, the compounds inhibited the LpxA enzymatic reaction in vitro. Intriguingly, using biochemical, biophysical, and structural characterization, we reveal two distinct mechanisms of LpxA inhibition; compound 1 is a substrate-competitive inhibitor targeting apo LpxA and compound 2 is an uncompetitive inhibitor targeting the LpxA-product complex. Compound 2 exhibited more favorable biological and physicochemical properties than compound 1, and was optimized using structural information to achieve improved antibacterial activity against wild type E. coli. These results show that LpxA is a promising antibacterial target and imply the advantages of targeting enzyme-product complexes in drug discovery.

Background Pseudomonas aeruginosa (PA) pneumonia is associated with morbidity and mortality in mechanically ventilated, intensive care unit (MV ICU) patients despite best clinical care. We assessed efficacy, PK, and safety of MEDI3902 in MV ICU subjects in the placebo-controlled, randomized Phase 2 EVADE study (NCT02696902; EudraCT 2015-001706-34). Methods Subjects with PCR-confirmed PA colonization of the lower respiratory tract were randomized to either a single IV infusion of 1,500 mg MEDI3902 (n = 85) or placebo (n = 83). Primary Efficacy endpoint was Endpoint Adjudication Committee-determined relative risk reduction (RRR) of PA pneumonia incidence in MEDI3902 vs. placebo recipients within 21 days post dose (2-sided α = 0.2). Serum MEDI3902 PK levels were measured through 49 days post dose. Treatment-emergent adverse events (TEAEs) and serious AEs (SAEs) were assessed through 49 days post dose. Results Baseline characteristics were similar between groups. MEDI3902 did not meet the primary endpoint of PA pneumonia vs. placebo (22.4% vs. 18.1%; RRR -23.7%, P = 0.491). Mean serum MEDI3902 level was 9.46 µg/mL (target 1.7µg/mL) at 21 days post dose, with a t½ 5.6 days. Proportion of subjects with TEAEs was similar between groups: ≥1 TEAE (98.8% MEDI3902; 97.6% placebo); ≥1 serious; and/or ≥grade 3 severity SAE (70.6% MEDI3902; 66.3% placebo). Deaths were numerically higher, although not statistically significant (24 (28.2%) MEDI3902 vs 19 (22.9%) Placebo; RRR -23.3%, P 0.429). Post-hoc analyses suggested RRR 47% among ~70% of the study population who had baseline Procalcitonin levels < 0.55 µg/L (12.5% MEDI3902 vs 23.7% placebo; 80%CI 6.1%-69.9%; P 0.135). Similarly, RRR 83% was observed among 50% of study subjects with baseline absolute neutrophil count (ANC) of < 8170 /µL (2.8% MEDI3902 vs 17.0% placebo; 80%CI 39.5%-95.5%; P 0.038). Subjects with Procalcitonin < 0.55 µg/L and ANC < 8170/ µL also had higher serum PK exposure. Conclusion A single IV dose of MEDI3902 provided PK exposure above the target level but did not achieve primary efficacy endpoint of reduction in PA pneumonia. Efficacy trends were observed in subjects with lower levels of baseline inflammatory biomarkers. MEDI3902 may have a path forward in certain patient populations such as ICU patients with lower baseline inflammation. Disclosures Jean Chastre, MD, AstraZeneca (Scientific Research Study Investigator) Marc Bourgeois, MD, AstraZeneca (Scientific Research Study Investigator) Apostolos Komnos, MD, PhD, AstraZeneca (Scientific Research Study Investigator) Ricard Ferrer, MD, PhD, Shionogi B.V. (Advisor or Review Panel member) Galia Rahav, MD, AstraZeneca (Scientific Research Study Investigator) Nicolas De Schryver, MD, AstraZeneca (Scientific Research Study Investigator) Alain Lepape, MD, AstraZeneca (Scientific Research Study Investigator) Miguel Sanchez Garcia, MD, PhD, AstraZeneca (Scientific Research Study Investigator) Antoni Torres, MD, PhD, AstraZeneca (Scientific Research Study Investigator) Omar Ali, PhD, AstraZeneca (Employee) Kathryn Shoemaker, MS, AstraZeneca (Employee) Alexey Ruzin, PhD, AstraZeneca (Employee, Shareholder) Yu Jiang, PhD, AstraZeneca (Employee) Susan Colbert, BSN, AstraZeneca (Employee) Drieke Vandamme, PhD, AstraZeneca (Scientific Research Study Investigator) Terramika Bellamy, n/a, AstraZeneca (Employee) Colin Reisner, MD, AstraZeneca (Employee) Filip Dubovsky, MD, MPH, AstraZeneca (Employee) Hasan S. Jafri, MD, FAAP, AstraZeneca (Employee)

Background Suvratoxumab is a human monoclonal antibody that neutralizes S. aureus (SA) alpha toxin (AT). SAATELLITE, a phase 2 study of the safety and efficacy of suvratoxumab for reducing the incidence of SA pneumonia (NCT02296320), was conducted within the consortium for Combatting Bacterial Resistance in Europe. Methods A total of 304 SA isolates (baseline, onset and last available isolates from suspected serious bacterial infections, SSBIs) collected from the lower respiratory tract samples from 165 subjects during SAATELLITE were subjected to whole genome sequencing. AT gene (hla) sequences were translated and amino acid variation was identified in comparison to the reference SA USA300 FPR3757. Phylogenetic analysis, genomic annotation and ST analysis were performed. AT expression in SA culture supernatants was performed by ELISA. Representative isolates with novel AT subtypes that had not been identified in previous studies were tested for hemolytic activity and suvratoxumab neutralizing activity. Wilcoxon rank sum test and Fisher’s exact test were performed, respectively: a) to compare difference in baseline AT expression in relation to SA pneumonia incidence; b) to evaluate the association between occurrence of AT stop codons and incidence of SA pneumonia at baseline, as well as the association between occurrence of AT stop codons and treatment arms at post baseline. Results We identified a total of 44 sequence types (STs) and 21 unique AT subtypes, 7 of which have not been described previously. No substitutions were located in the suvratoxumab binding region and all novel AT subtypes displaying lytic activity were neutralized by suvratoxumab. We detected stop codons Q113B and W205B in AT sequences in 53 and 2 SA isolates, respectively. We uncovered no significant associations of: 1) baseline AT expression with SA pneumonia incidence [p=0.967]; 2) occurrence of AT gene stop codon with either SA pneumonia incidence [p >0.999] or suvratoxumab treatment [p=0.103; lower frequency of stop codons in suvratoxumab arm versus placebo]. Conclusion Our data indicated that: 1) suvratoxumab target region in (AT) remains conserved; 2) suvratoxumab is active against all AT variants identified to date; 3) suvratoxumab did not exert pressure on SA clinical isolates for selection of escape mutants. Disclosures David E. Tabor, PhD, AstraZeneca (Employee, Shareholder) Andrey Tovchigrechko, PhD, AstraZeneca (Employee, Shareholder)KitePharma, a Gilead company (Employee, Shareholder) Bret R. Sellman, PhD, AstraZeneca (Employee, Shareholder) Michael McCarthy, n/a, AstraZeneca (Employee) Kathryn Shoemaker, MS, AstraZeneca (Employee) Hasan S. Jafri, MD, FAAP, AstraZeneca (Employee) Mark T. Esser, PhD, AstraZeneca (Employee) Alexey Ruzin, PhD, AstraZeneca (Employee, Shareholder)

Background Suvratoxumab (suvra), an extended half-life (~80 days), Staphylococcus aureus (SA) alpha toxin-neutralizing IgG monoclonal antibody, is under investigation for prevention of SA pneumonia in patients on mechanical ventilation (MV). We characterized the serum PK of suvra using population pharmacokinetics (popPK) in both healthy volunteers and MV patients and quantified the proportion of patients reaching the serum target of 211 μg/mL at 30 days post-dose. Methods The popPK analysis included 1,368 serum samples from two early phase studies (NCT02296320; EudraCT 2014-001097-34): (1) Phase 1 study in 26 healthy adults receiving single IV suvra doses ranging from 0.225g to 5g, with PK sampled up to 360 days; and (2) Phase 2 study in MV patients with PCR-confirmed SA colonization of lower respiratory tract receiving one suvra IV dose of 2g (n = 15) or 5g (n = 96), with PK sampled up to 100 days. Results A two-compartment linear model with weight-based scaling of the PK parameters adequately described the serum PK data (Figure 1). MV status, number of days on MV, and age impacted the PK of suvra. A moderate between-subject variability ( Conclusion MV status, post-dose duration on MV, body weight, and age were identified as statistically significant covariates influencing the PK of suvra. Serum PK and popPK analyses support the 5g dose for future studies with suvra in MV patients. Disclosures All authors: No reported disclosures.

Introduction Staphylococcus aureus (S. aureus) is a common cause of ventilator-associated pneumonia. Rapid and accurate detection of lower respiratory tract colonization and/or infection with S. aureus may inform targeted preventive and therapeutic strategies. To investigate this, we compared semi-quantitative (SQ)-culture results from 79 endotracheal aspirates (ETA) collected from mechanically-ventilated patients, to two culture and two non-culture-based methods for detection of S. aureus. Methods ETA analyzed by routine SQ-culture on blood and colistin-nalidixic-acid agar was compared to: (i) quantitative (Q-) culture on chromogenic COLOREX™ Staph aureus; (ii) enrichment in brain-heart-infusion broth followed by plating on blood agar and COLOREX™; (iii) nuc-based TaqMan qPCR, and (iv) GeneXpert MRSA/SA ETA assay. Results Of the 79 ETA samples analyzed by SQ-culture, 39 samples were positive, and 40 negative for S. aureus. Two samples negative for S. aureus by SQ-culture were, however, S. aureus-positive by the other four methods and were considered positive. Appending these two samples as positive in the SQ-culture results, sensitivities−specificities for Q-culture, enrichment-culture, TaqMan qPCR and GeneXpert were 100–95, 100–92, 100–53% and 100% − 100, respectively. The lower specificities of Q-culture, enrichment-culture, and TaqMan qPCR was because of their higher sensitivities, although TaqMan qPCR also detected S. aureus-specific extracellular DNA. Conclusion This first evaluation of the GeneXpert MRSA/SA ETA assay with ETA samples found it to be highly sensitive, specific, user-friendly (hands-on time ~ 5 min.), and rapid (~ 66 min. assay time). Where this equipment is not available, we recommend implementing more sensitive culture-based methods for improved S. aureus detection in ETA samples. Electronic supplementary material The online version of this article (10.1186/s13756-018-0460-8) contains supplementary material, which is available to authorized users.

Tight coordination of inner and outer membrane biosynthesis is very important in Gram-negative bacteria. Biosynthesis of the lipid A moiety of lipopolysaccharide, which comprises the outer leaflet of the outer membrane has garnered interest for Gram-negative antibacterial discovery. In particular, several potent inhibitors of LpxC (the first committed step of the lipid A pathway) are described. Here we show that serial passaging of Klebsiella pneumoniae in increasing levels of an LpxC inhibitor yielded mutants that grew only in the presence of the inhibitor. These strains had mutations in fabZ and lpxC occurring together (encoding either FabZR121L/LpxCV37G or FabZF51L/LpxCV37G). K. pneumoniae mutants having only LpxCV37G or LpxCV37A or various FabZ mutations alone were less susceptible to the LpxC inhibitor and did not require LpxC inhibition for growth. Western blotting revealed that LpxCV37G accumulated to high levels, and electron microscopy of cells harboring FabZR121L/LpxCV37G indicated an extreme accumulation of membrane in the periplasm when cells were subcultured without LpxC inhibitor. Significant accumulation of detergent-like lipid A pathway intermediates that occur downstream of LpxC (e.g., lipid X and disaccharide monophosphate [DSMP]) was also seen. Taken together, our results suggest that redirection of lipid A pathway substrate by less active FabZ variants, combined with increased activity from LpxCV37G was overdriving the lipid A pathway, necessitating LpxC chemical inhibition, since native cellular maintenance of membrane homeostasis was no longer functioning. IMPORTANCE Emergence of antibiotic resistance has prompted efforts to identify and optimize novel inhibitors of antibacterial targets such as LpxC. This enzyme catalyzes the first committed step of lipid A synthesis, which is necessary to generate lipopolysaccharide and ultimately the Gram-negative protective outer membrane. Investigation of this pathway and its interrelationship with inner membrane (phospholipid) biosynthesis or other pathways is therefore highly important to the fundamental understanding of Gram-negative bacteria and by extension to antibiotic discovery. Here we exploited the availability of a novel LpxC inhibitor to engender the generation of K. pneumoniae resistant mutants whose growth depends on chemical inhibition of LpxC. Inhibitor dependency resulted from the interaction of different resistance mutations and was based on loss of normal cellular mechanisms required to establish membrane homeostasis. This study provides new insights into the importance of this process in K. pneumoniae and how it may be linked to novel biosynthetic pathway inhibitors.

Atopic dermatitis (AD) is a chronic inflammatory disease with significant local and systemic inflammation and barrier disruption. AD is associated with increased risk of allergen sensitization and skin colonization by Staphylococcus aureus. The heterogeneity of AD is unknown, and its complexity suggests its subdivision into several endotypes.To evaluate allergy-driven endotypic differences in patients with AD and identify proteomic signatures to distinguish between inflammatory responses. To perform proteomic profiling of allergen sensitivity, antibody levels to S aureus antigens, and circulating inflammatory mediators to characterize AD subsets in 76 subjects with moderate to severe AD and 39 healthy controls (HCs).Sera were collected from 76 subjects with moderate to severe AD and 39 HCs with no history of skin disease. Serum was tested for levels of total serum immunoglobulin E (IgE) and allergen-specific IgE using a panel of 119 allergens as well as IgE antibodies against S aureus antigens, and was profiled for more than 1100 proteins by SOMAscan to detect differential expression of inflammatory mediators.Total serum IgE levels were significantly (P.001) elevated in subjects with AD versus controls. A greater percentage of subjects with AD were allergic compared with HCs, and patients with AD tested positive to a greater number of allergens than did HCs. IgE was upregulated across 4 allergen subsets (food, perennial, seasonal, and mixed), and each allergen subset was associated with a distinct inflammatory signature marked by a specific suite of upregulated proteins. Finally, IgE antibodies against S aureus toxic shock syndrome toxin-1 were significantly upregulated in subjects with seasonal allergy (P = .0430) and perennial allergy (P = .00032).Overall, this study addresses the heterogeneity of AD by characterizing subsets of AD on the basis of allergen sensitization. It also demonstrates the differential systemic inflammation and S aureus-specific antibody responses associated with the allergenic endotypes. These unique proteomic signatures may be valuable for precise disease characterization and subsequent personalized treatment.

The monobactam scaffold is attractive for the development of new agents to treat infections caused by drug-resistant Gram-negative bacteria because it is stable to metallo-β-lactamases (MBLs). However, the clinically used monobactam aztreonam lacks stability to serine β-lactamases (SBLs) that are often coexpressed with MBLs. LYS228 is stable to MBLs and most SBLs. LYS228 bound purified Escherichia coli penicillin binding protein 3 (PBP3) similarly to aztreonam (derived acylation rate/equilibrium dissociation constant [k(2)/K(d)] of 367,504 s(−1) M(−1) and 409,229 s(−1) M(−1), respectively) according to stopped-flow fluorimetry. A gel-based assay showed that LYS228 bound mainly to E. coli PBP3, with weaker binding to PBP1a and PBP1b. Exposing E. coli cells to LYS228 caused filamentation consistent with impaired cell division. No single-step mutants were selected from 12 Enterobacteriaceae strains expressing different classes of β-lactamases at 8× the MIC of LYS228 (frequency

LYS228 is a novel monobactam with potent activity against Enterobacteriaceae. LYS228 is stable to metallo-β-lactamases (MBLs) and serine carbapenemases, including Klebsiella pneumoniae carbapenemases (KPCs), resulting in potency against the majority of extended-spectrum β-lactamase (ESBL)-producing and carbapenem-resistant Enterobacteriaceae strains tested. Overall, LYS228 demonstrated potent activity against 271 Enterobacteriaceae strains, including multidrug-resistant isolates. Based on MIC(90) values, LYS228 (MIC(90), 1 μg/ml) was ≥32-fold more active against those strains than were aztreonam, ceftazidime, ceftazidime-avibactam, cefepime, and meropenem. The tigecycline MIC(90) was 4 μg/ml against the strains tested. Against Enterobacteriaceae isolates expressing ESBLs (n = 37) or displaying carbapenem resistance (n = 77), LYS228 had MIC(90) values of 1 and 4 μg/ml, respectively. LYS228 exhibited potent bactericidal activity, as indicated by low minimal bactericidal concentration (MBC) to MIC ratios (MBC/MIC ratios of ≤4) against 97.4% of the Enterobacteriaceae strains tested (264/271 strains). In time-kill studies, LYS228 consistently achieved reductions in CFU per milliliter of 3 log(10) units (≥99.9% killing) at concentrations ≥4× MIC for Escherichia coli and K. pneumoniae reference strains, as well as isolates encoding TEM-1, SHV-1, CTX-M-14, CTX-M-15, KPC-2, KPC-3, and NDM-1 β-lactamases.

Background Bispecific antibody MEDI3902, targeting the Pseudomonas aeruginosa type 3 secretion system (PcrV) and Psl exopolysaccharide, is currently in phase 2b development for prevention of nosocomial pneumonia in patients undergoing mechanical ventilation. We surveyed a diverse collection of isolates to study MEDI3902 epitope conservation and protective activity. Methods P. aeruginosa clinical isolates (n = 913) were collected from diverse patients and geographic locations during 2003-2014. We conducted whole-genome sequencing; performed PcrV and Psl expression analyses via immunoblotting and enzyme-linked immunosorbent assay, respectively; performed crystallography to determine the MEDI3902 PcrV epitope, using anti-PcrV Fab and PcrV components (resolved at 2.8 A); and evaluated MEDI3902 protective activity against select isolates in vitro and in vivo. Results Intact psl operon and pcrV genes were present in 94% and 99% of isolates, respectively, and 99.9% of isolates contained at least one of the genetic elements. Anti-Psl binding was confirmed in tested isolates harboring a complete Psl operon or lacking nonessential psl genes. We identified 46 PcrV variant sequences, and MEDI3902-PcrV contact residues were preserved. MEDI3902 maintained potent in vivo activity against various strains, including strains expressing only a single target. Conclusions Psl and PcrV are highly prevalent in global clinical isolates, suggesting MEDI3902 can mediate broad coverage against P. aeruginosa.

Mechanical ventilation (MV) is the primary risk factor for the development of ventilator-associated pneumonia (VAP). Besides inducing a pro-inflammatory T-helper (Th)-1 cytokine response, MV also induces an anti-inflammatory Th2 cytokine response, marked by increased IL-4 secretion and reduced bacterial phagocytic capacity of rodent lung macrophages. Since IL-4 is known to downregulate both Th1 and Th17 cytokines, the latter is important in mediating mucosal immunity and combating bacterial and fungal growth, we studied and showed here in a rat model of MV that Th17 cytokines (IL-17A, IL-17F, and IL-22) were significantly upregulated in the lung as a response to different MV strategies currently utilized in clinic. To study whether the increased IL-4 levels are associated with downregulation of the anti-bacterial Th17 cytokines, we subsequently challenged mechanically ventilated rats with an intratracheal inoculation of Pseudomonas aeruginosa (VAP model) and showed a dramatic downregulation of IL-17A, IL-17F, and IL-22, compared to animals receiving the same bacterial burden without MV. For the studied Th1 cytokines (IFNγ, TNFα, IL-6, and IL-1β), only IFNγ showed a significant decrease as a consequence of bacterial infection in mechanically ventilated rats. We further studied IL-17A, the most studied IL-17 family member, in intensive care unit (ICU) pneumonia patients and showed that VAP patients had significantly lower levels of IL-17A in the endotracheal aspirate compared to patients entering ICU with pre-existing pneumonia. These translational data, obtained both in animal models and in humans, suggest that a deficient anti-bacterial Th17 response in the lung during MV is associated with VAP development.

Background Patients with lower airway Staphylococcus aureus (SA) colonization are at great risk (> 20%) of early-onset ventilator-associated pneumonia (VAP). Thus, a rapid test is required to identify patients at risk. Suvratoxumab (formerly MEDI4893) is a human monoclonal antibody that neutralizes SA alpha toxin. SAATELLITE, a phase 2 study of safety and efficacy of suvratoxumab for reducing the incidence of SA pneumonia (NCT02296320) was conducted and recently completed within the consortium for Combatting Bacterial Resistance in Europe. We investigated the performance of a rapid PCR test (Xpert MRSA/SA SSTI™, Cepheid) as a screening tool during the study and the association between SA load and suvratoxumab efficacy. Methods The PCR assay was used to detect SA and methicillin-resistant SA (MRSA) in lower respiratory tract (LRT) samples. Culture was performed on PCR SA+ LRT samples according to local procedures. PCR SA+ subjects were randomized 1:1 to either a single intravenous infusion of 5000 mg suvratoxumab (n = 96) or placebo (n = 100) and followed for 190 days post dose. Efficacy of suvratoxumab was defined as relative risk reduction (RRR) in incidence of SA pneumonia within 30 days post-dose compared with placebo. Results 299 (41.5%) out of 720 screened subjects were SA+ by PCR. Of 209 subjects with culture data, there were 162 (77.5%) SA+, 47 (22.5%) SA- and 9 (5.6%) MRSA by culture. Culture results could have been affected by antibiotic use and site variability in limits of detection ranging from 3.3 to 100,000 colony-forming units per mL (CFU/mL). No discordance was noted between PCR and culture for MRSA detection. An inverse linear correlation was observed between the PCR cycle threshold (Ct) values for SA protein A gene (spa) and SA CFU/mL counts from quantitative culture. In subjects with low SA load (Ct ≥ 29; n = 72), suvratoxumab provided 66.7% RRR [90% confidence interval (CI): 21.3%, 86.2%] compared with 31.9% RRR [90% CI: -7.5%, 56.8%] in total study population. Conclusion Cepheid Xpert PCR assay was easy to perform, sensitive and standardized, and provided better sensitivity than conventional culture for detection of SA. Additionally, quantitative PCR Ct output was associated with the efficacy of suvratoxumab in reducing SA pneumonia incidence. Disclosures All authors: No reported disclosures.

Background Staphylococcus aureus (SA) pneumonia imposes significant morbidity and mortality in mechanically ventilated, intensive care unit (MV ICU) patients despite best clinical care. We assessed efficacy, PK, AT-neutralizing antibodies (AT NAbs), and safety of suvratoxumab (suvra) in MV ICU subjects in the placebo-controlled, randomized Phase 2 SAATELLITE study (NCT02296320; EudraCT 2014-001097-34). Methods Subjects with PCR-confirmed SA colonization of the lower respiratory tract were randomized to either a single intravenous infusion of 5,000 mg suvra (n = 96) or placebo (n = 100) and followed for 190 days post dose. Efficacy endpoints were Endpoint Adjudication Committee-determined relative risk reduction (RRR) of SA pneumonia incidence in suvra vs. placebo recipients within 30 days post dose (primary endpoint, tested at 2-sided α = 0.1), incidence of all-cause pneumonia, and all-cause pneumonia or death. Serum suvra PK and levels of AT NAbs were measured through 90 days post dose and analyzed for statistical correlation. Treatment-emergent adverse events (TEAEs) and serious AEs (SAEs) were assessed through 190 days post dose. Results Baseline characteristics were similar between groups. Suvra provided 31.9% RRR in incidence of SA pneumonia vs. placebo (17.7% vs. 26%; P = 0.166), 30% RRR (P = 0.146) in incidence of all-cause pneumonia, and 23% RRR (P = 0.164) in incidence of all-cause pneumonia or death. Suvra reduced mean hospital stay and ICU duration by 3.0 and 2.4 days, resp. vs. placebo. Mean serum ± SD suvra level was 296 ± 131 µg/mL at 30 days post dose. Serum AT Nab ± SD levels reached 156.03 ± 72.48 IU/mL at 2 days post dose, declining slowly to 33.74 ± 16.04 IU/mL by 90 days post dose. AT NAbs correlated with PK (r2 = 0.7), thereby confirming functional activity of suvra over time. Proportion of subjects with TEAEs or SAEs was similar between groups: ≥1 TEAE (93.8% suvra; 93.0% placebo); ≥1 serious; and/or ≥grade 3 severity SAE (66.7% suvra; 58.0% placebo). Conclusion A single intravenous dose of suvra produced a trend toward reduced incidence of SA pneumonia, health resource savings, sustained functional exposure in serum, and an acceptable safety profile. These results support continued development of suvra in MV ICU patients. Disclosures All Authors: No reported Disclosures.

Staphylococcus aureus causes an array of serious infections resulting in high morbidity and mortality worldwide. This study evaluated naturally occurring serum anti-alpha-toxin (anti-AT) antibody levels in human subjects from various age groups, individuals with S. aureus dialysis and surgical-site infections, and S. aureus -colonized versus noncolonized subjects. Anti-AT immunoglobulin G (IgG) and neutralizing antibody (NAb) levels in infants (aged ≤1 year) were significantly lower than those in other populations. In comparison to adolescent, adult, and elderly populations, young children (aged 2 to 10 years) had equivalent anti-AT IgG levels but significantly lower anti-AT NAb levels. Therefore, the development of anti-AT NAbs appears to occur later than that of AT-specific IgG, suggesting a maturation of the immune response to AT. Anti-AT IgG levels were slightly higher in S. aureus -colonized subjects than in noncolonized subjects. The methicillin susceptibility status of colonizing isolates had no effect on anti-AT antibody levels in S. aureus -colonized subjects. The highest anti-AT IgG and NAb levels were observed in dialysis patients with acute S. aureus infection. Anti-AT IgG and NAb levels were well correlated in subjects aged >10 years, regardless of colonization or infection status. These data demonstrate that AT elicits a robust IgG humoral response in infants and young children that becomes stable prior to adolescence, matures into higher levels of NAbs in healthy adolescents, and becomes elevated during S. aureus infection. These findings may assist in identifying subjects and patient populations that could benefit from vaccination or immunoprophylaxis with anti-AT monoclonal antibodies.

Objectives: Current treatments for Clostridium difficile infection include vancomycin, metronidazole, and fidaxomicin. LFF571 is an experimental agent undergoing evaluation in humans for the treatment of moderate C. difficile infection. Reduced susceptibility of C. difficile to fidaxomicin or LFF571 in vitro can be mediated by single point mutations in the genes encoding the targets, whereas the mechanism(s) mediating reduced susceptibility to vancomycin in vitro remains elusive. To further characterize mechanisms reducing susceptibility of C. difficile to vancomycin, fidaxomicin, or LFF571 in vitro selections via serial passage at low cell density and whole genome sequencing were performed. Methods: C. difficile strains ATCC43255 and three clinical isolates were subjected to 10 passages on medium containing a range of concentrations of fidaxomicin, LFF571 or vancomycin. Genomic DNA from isolates with reduced susceptibility was sequenced using Illumina Whole Genome Sequencing. Results: Clones exhibiting decreased susceptibility to fidaxomicin harbored mutations in rpoB and CD22120 (marR homolog). Clones exhibiting decreased susceptibility to vancomycin harbored mutations in rpoC and also in CD2725, CD3659 and sdaB which encode putative N-acetylglucosamine transferase, exonuclease and L-serine deaminase, respectively. All mutations resulted in nonsynonymous substitutions in the encoded proteins. No clones with reduced susceptibility to LFF571 were selected in this study. Conclusions: Reduced susceptibility to fidaxomicin and vancomycin was associated with mutations mediating target modifications (RNA polymerase or cell wall, respectively), as well as with mutations that may contribute to reduced susceptibility via other mechanisms. The MIC of LFF571 was unaffected in those mutants with reduced susceptibility to fidaxomicin or vancomycin.

BACKGROUND:Respiratory syncytial virus (RSV) is an established cause of serious lower respiratory disease in infants, elderly and high-risk populations. The OUTSMART surveillance program aims to characterize patient populations and currently circulating RSV strains, and monitor temporal and geographic evolution of RSV F and G proteins in the U.S. METHODS:The OUTSMART 2016-17 study collected RSV-positive samples from 25 RSVAlert® laboratories from 4 U.S. regions and Puerto Rico during November 2016 through March 2017. Frequencies of A and B subtypes and genotypes were determined for several demographic and geographic variables. To gauge the representativeness of the OUTSMART patients, results were compared to discharge data from the NEDS and NIS databases. RESULTS:A total of 1,041 RSV-positive samples with associated demographic data were obtained and the RSV F gene and second variable region of the G gene were sequenced. The majority of samples (76.0%) came from children under 2 years old: 24 hrs) occurred in 29.0% of patients of which 52.0% had RSV B. Outpatients (LOS

Objectives MEDI4893 is a novel, long‐acting human monoclonal antibody targeting Staphylococcus aureus (SA) alpha toxin (AT). This report presents the results of the exploratory analyses from a randomised phase 1 dose‐escalation study in healthy human subjects receiving single intravenous MEDI4893 doses or placebo. Methods Anti‐AT antibodies and AT expression were measured as described previously. Nasal swabs were analysed by culture and PCR. Data were summarised by treatment groups and visits by using SAS System Version 9.3. Results Subjects receiving 2250 or 5000 mg of MEDI4893 had the highest serum anti‐AT neutralising antibody (NAb) levels: approximately 180‐ to 240‐, 70‐ to 100‐ and sevenfold to 10‐fold higher than respective baseline levels at peak, 30 and 360 days, respectively. In these subjects, levels of serum anti‐AT NAbs were >3.2 International Units (IU) mL−1 for at least 211 days. In the upper respiratory tract, anti‐AT NAb levels increased with MEDI4893 dose. No apparent effect of MEDI4893 on SA nasal colonisation, hla gene sequence or AT expression was observed. Five AT variants were detected, their lytic activity was fully neutralised by MEDI4893. Discussion Our results indicate that (1) MEDI4893 administration at 2250 and 5000 mg would provide effective immunoprophylaxis against systemic SA disease; (2) MEDI4983 distributes to the upper respiratory tract and retains neutralising activity against AT; and (3) potential for emergence of MEDI4893 resistance is low. Conclusion Intravenous administration of MEDI4893 maintained levels of anti‐AT NAbs in serum and nasal mucosa that may provide effective immunoprophylaxis against SA disease and support continued clinical development of MEDI4893.

TEM- and SHV-type extended-spectrum β-lactamases (ESBLs) are the most common ESBLs found in the United States and are prevalent throughout the world. Amino acid substitutions at a number of positions in TEM-1 lead to the ESBL phenotype, although substitutions at residues 104 (E to K), 164 (R to S or H), 238 (G to S), and 240 (E to K) appear to be particularly important in modifying the spectrum of activity of the enzyme. The SHV-1-derived ESBLs are a less diverse collection of enzymes; however, the majority of amino acid substitutions resulting in an ESBL mirror those seen in the TEM-1-derived enzymes. Pyrosequencing by use of the single-nucleotide polymorphism (SNP) protocol was applied to provide sequence data at positions critical for the ESBL phenotype spanning the bla TEM and bla SHV genes. Three novel β-lactamases are described: the ESBLs TEM-155 (Q39K, R164S, E240K) and SHV-105 (I8F, R43S, G156D, G238S, E240K) and a non-ESBL, SHV-48 (V119I). The ceftazidime, ceftriaxone, and aztreonam MICs for an Escherichia coli isolate expressing bla SHV-105 were >128, 128, and >128 μg/ml, respectively. Likewise, the ceftazidime, ceftriaxone, and aztreonam MICs for an E. coli isolate expressing bla TEM-155 were >128, 64, and > 128 μg/ml, respectively. Pyrosequence analysis determined the true identity of the β-lactamase on plasmid R1010 to be SHV-11 rather than SHV-1, as previously reported. Pyrosequencing is a real-time sequencing-by-synthesis approach that was applied to SNP detection for TEM- and SHV-type ESBL identification and represents a robust tool for rapid sequence determination that may have a place in the clinical setting.

In concert with the development of novel β-lactams and broad-spectrum cephalosporins, bacterially encoded β-lactamases have evolved to accommodate the new agents. This study was designed to identify, at the sequence level, the genes responsible for the extended-spectrum-β-lactamase (ESBL) phenotypes of Escherichia coli, Klebsiella pneumoniae , and Proteus mirabilis isolates collected during the global tigecycline phase 3 clinical trials. PCR assays were developed to identify and clone the bla TEM , bla SHV , bla OXA , and bla CTX genes from clinical strains. Isolates were also screened for AmpC genes of the bla CMY , bla ACT , bla FOX , and bla DHA families as well as the bla KPC genes encoding class A carbapenemases. E. coli, K. pneumoniae , and P. mirabilis isolates with ceftazidime MICs of ≥2 μg/ml were designated possible ESBL-producing pathogens and were then subjected to a confirmatory test for ESBLs by use of Etest. Of 272 unique patient isolates, 239 were confirmed by PCR and sequencing to carry the genes for at least one ESBL, with 44% of the positive isolates harboring the genes for multiple ESBLs. In agreement with current trends for ESBL distribution, bla CTX-M -type β-lactamase genes were found in 83% and 71% of the ESBL-positive E. coli and K. pneumoniae isolates, respectively, whereas bla SHV genes were found in 41% and 28% of the ESBL-positive K. pneumoniae and E. coli isolates, respectively. Ninety-seven percent of the E. coli and K. pneumoniae isolates were tigecycline susceptible (MIC 90 = 2 μg/ml), warranting further studies to define the therapeutic utility of tigecycline against strains producing ESBLs in a clinical setting.

Objectives The purpose of this study was to characterize decreased susceptibility to tigecycline in clinical isolates of Escherichia coli obtained during Phase 3 clinical trials. Methods Gene expression was analysed by transcriptional profile analysis and RT-PCR. Transposon mutagenesis with IS903kan was used for selection of transposon mutants. Transposon insertions were mapped by DNA sequencing and PCR analyses. The MICs were determined by broth microdilution. Results Both transcriptional profile analysis and Taqman RT-PCR demonstrated increased expression levels of MarA, a transcriptional activator, and AcrAB, an RND-type efflux pump, in the strains with elevated tigecycline MICs. Transposon mutagenesis generated nine mutants, the majority of which had either marA or acrB inactivated. Sequence analysis revealed a single nucleotide insertion in the open reading frame of the marR gene in less-susceptible strains of E. coli. Conclusions This study suggested that a loss of MarR functionality due to a frameshift mutation resulted in constitutive overproduction of MarA and AcrAB and, consequently, in decreased susceptibility to tigecycline in clinical isolates of E. coli.

Tigecycline, a novel broad-spectrum glycylcycline antibiotic, is active against many gram-positive and gram-negative bacterial pathogens including most strains of Enterobacter cloacae. Recently, however, a few clinical strains of E. cloacae with decreased susceptibility to tigecycline were isolated. In this study, two tigecycline-susceptible mutants of E. cloacae, GC7696 and GC7697, were obtained by transposon mutagenesis of a tigecycline-resistant clinical isolate G946. Transposon insertions were mapped to either the acrA or acrB genes. Restoration of the original resistant phenotype occurred when GC7696 and GC7697 were transcomplemented with a plasmid harboring the intact acrAB region amplified from G946. Northern blot analysis of G946 and several other E. cloacae clinical strains that exhibited decreased susceptibility to tigecycline, revealed increased levels of the acrAB transcript. In addition, overexpression of acrAB correlated with increased expression of the ramA gene, whereas the expression of another transcriptional activator, marA, was not changed. These results suggest that decreased susceptibility to tigecycline in E. cloacae is the result of RamA-mediated overexpression of the AcrAB efflux pump.

Previous studies suggested that a Gly-containing branch of cell wall precursor [C 55 -MurNAc-(peptide)-GlcNAc], which is often referred to as lipid II, might serve as a nucleophilic acceptor in sortase-catalyzed anchoring of surface proteins in Staphylococcus aureus. To test this hypothesis, we first simplified the procedure for in vitro biosynthesis of Gly-containing lipid II by using branched UDP-MurNAc-hexapeptide isolated from the cytoplasm of Streptomyces spp. Second, we designed a thin-layer chromatography-based assay in which the mobility of branched but not linear lipid II is shifted in the presence of both sortase and LPSTG-containing peptide. These results and those of additional experiments presented in this study further suggest that lipid II indeed serves as a natural substrate in a sorting reaction.

This chapter summarizes specific resistance mechanisms found in the staphylococci. When discussing resistance among the staphylococci, it is important to draw a distinction between community-acquired versus hospital-acquired (nosocomial) infections. Strains resistant to arsenicals and mercury were identified well before what is now known as the antibiotic era. From the genetic point of view, resistance falls into one of two classes: mutation of a bacterial gene or acquisition of a dedicated resistance gene from some other organism by some form of genetic exchange (transduction, conjugation, or transformation). In the history of antimicrobial chemotherapy, the most useful of antistaphylococcal agents have been the beta-lactam antibiotics, the prototype of which is penicillin. These agents, which include several structural classes, all contain one common structural feature: the beta-lactam ring. Rifampin, a member of the rifamycin class of antibiotics, inhibits transcription by attacking the beta-subunit of RNA polymerase. The fluoroquinolone antimicrobials are one of the few classes of antibacterial agents that are not based on a natural product. Sulfonamide resistance in the staphylococci, which arose soon after the introduction of the sulfa drugs, is chromosomally encoded (by the sulA gene) and is attributed to the overproduction of p-aminobenzoate. Mupirocin (formulated with the trade name Bactroban) has come into wide use as a topical agent for the treatment of gram-positive infections and more recently has been employed successfully to treat nasal carriers of methicillin-resistant S. aureus (MRSA), especially those in chronic care settings (e.g., nursing homes) and hospital staff.

The Staphylococcus aureus gene for toxic shock toxin (tst) is carried by a 15 kb mobile pathogenicity island, SaPI1, that has an intimate relationship with temperate staphylococcal phage 80alpha. During phage growth, SaPI1 is excised from its unique chromosomal site, attC, replicates autonomously, interferes with phage growth, and is efficiently encapsidated into special small phage heads commensurate with its size. Upon transfer to a recipient organism, SaPI1 integrates at attC by means of a self-coded integrase. One or more phage functions are required for excision, autonomous replication and encapsidation of the element and, thus, the overall relationship between SaPI1 and 80alpha is similar to that between coliphages P4 and P2. Among other staphylococcal phages tested, only phi13 interacts with SaPI1, inducing excision but not replication or transfer of the element.

The relationship between expression of adeABC and minimal inhibitory concentration (MIC) of tigecycline was investigated by RT-PCR and statistical analyses in a population of 106 clinical isolates (MIC range, 0.0313-16 microg/ml) of Acinetobacter calcoaceticus-Acinetobacter baumannii complex. There was a statistically significant linear relationship (p0.0001) between log-transformed expression values and log-transformed MIC values, indicating that overexpression of AdeABC efflux pump is a prevalent mechanism for decreased susceptibility to tigecycline in A. calcoaceticus-A. baumannii complex.