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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.

Antibiotic resistance poses a global health threat, but the within-host drivers of resistance remain poorly understood. Pathogen populations are often assumed to be clonal within hosts, and resistance is thought to emerge due to selection for de novo variants. Here we show that mixed strain populations are common in the opportunistic pathogen P. aeruginosa . Crucially, resistance evolves rapidly in patients colonized by multiple strains through selection for pre-existing resistant strains. In contrast, resistance evolves sporadically in patients colonized by single strains due to selection for novel resistance mutations. However, strong trade-offs between resistance and growth rate occur in mixed strain populations, suggesting that within-host diversity can also drive the loss of resistance in the absence of antibiotic treatment. In summary, we show that the within-host diversity of pathogen populations plays a key role in shaping the emergence of resistance in response to treatment. Here, Caballero et al. provide an in depth characterisation of patients colonized with single or mixed strains of Pseudomonas aeruginosa to demonstrate the impact of within-host diversity on the development of antibiotic resistance.

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 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. 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. 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: <1 year (48.4%), 1-2 years (27.6%). The OUTSMART patient sample was similar to NEDS and NIS for age, gender, and geographic location. Both OUTSMART and national RSV cases peaked in January. Of OUTSMART samples, 45.3% were subtype A, 53.7% were subtype B and 1.0% were mixed A and B. The percentage of RSV B cases increased with increasing age. Hospitalization (length of hospital stay, LOS, >24 hrs) occurred in 29.0% of patients of which 52.0% had RSV B. Outpatients (LOS <24 hrs) were 64.4% of total of which 73.3% were diagnosed in the ER and discharged, while only 6% were diagnosed in other outpatient settings. The OUTSMART 2016-17 study was representative of the U.S. RSV experience. Geographic and temporal information from the RSV surveillance program will be used to establish a molecular baseline of RSV F and G sequence variability and to help inform development of novel agents for RSV prophylaxis and treatment.

Objectives 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. Methods 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. Results 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 mL−1 for SARS‐CoV‐2 spike (S), receptor‐binding domain (RBD) and nucleocapsid (N) antigens, respectively. For all antigens, analytical specificity was > 90% and clinical specificity was 99.0%. Clinical sensitivities for S, RBD and N antigens were 100%, 98.8% and 84.9%, respectively. Comparison with the Elecsys® immunoassay showed ≥ 87.7% agreement and linear correlation (Pearson r of 0.85, P < 0.0001) relative to the MN assay. Conversion factors for the WHO International Standard and Meso Scale Discovery® Reference Standard are presented. Conclusions The 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. 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. This 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.

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.

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. 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 FabZ R121L /LpxC V37G or FabZ F51L /LpxC V37G ). K. pneumoniae mutants having only LpxC V37G or LpxC V37A or various FabZ mutations alone were less susceptible to the LpxC inhibitor and did not require LpxC inhibition for growth. Western blotting revealed that LpxC V37G accumulated to high levels, and electron microscopy of cells harboring FabZ R121L /LpxC V37G 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 LpxC V37G 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.

Introduction Pseudomonas aeruginosa is a common cause of ventilator-associated pneumonia (VAP). Rapid and accurate detection of lower respiratory tract colonization and/or infection with P. aeruginosa may 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 of P. aeruginosa. Methods P. 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 ChromID P. aeruginosa and blood agar; (iii) enrichment in brain heart infusion broth followed by plating on blood agar and ChromID P. aeruginosa ; (iv) O-antigen acetylase gene-based TaqMan qPCR; and (v) GeneXpert PA PCR assay. Results Of the 80 ETA samples included, one sample that was negative for P. aeruginosa by 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. Conclusion This 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 for P. aeruginosa detection in ETA samples.

Objective To understand the relationships of Staphylococcus aureus (SA) bacteremic pneumonia (SABP) outcome with patient‐specific and SA‐specific variables. Methods We analysed SA bloodstream isolates and matching sera in SABP patients by sequencing SA isolates (n = 50) and measuring in vitro AT production, haemolytic activity and expression of ClfA and ClfB. Controls were sera from gram‐negative bacteremia patients with or without pneumonia and uninfected subjects. Levels of IgGs, IgMs and neutralizing antibodies (NAbs) against SA antigens were quantified and analysed by one‐way ANOVA. Associations of patient outcomes with patient variables, antibody levels and isolate characteristics were evaluated by univariate and multivariate logistic regression analyses. Results SABP patients had higher levels of IgGs against eight virulence factors and anti‐alpha toxin (AT) NAbs than uninfected controls. Levels of IgG against AT and IgMs against ClfA, FnbpA and SdrC were higher in clinically cured SABP patients than in clinical failures. Anti‐LukAB NAb levels were elevated in all cohorts. Increased odds of cure correlated with higher haemolytic activity of SA strains, longer time between surgery and bacteremia (> 30 days), longer duration of antibiotic therapy, lower acute physiology and total APACHE II scores, lack of persistent fever for > 72 h and higher levels of antibodies against AT (IgG), ClfA (IgM), FnbpA (IgM) and SdrC (IgM). Discussion Limitations included the cross‐sectional observational nature of the study, small sample size and inability to measure antibody levels against all SA virulence factors. Conclusion Our results suggest that SABP patients may benefit from immunotherapy targeting multiple SA antigens. Increased odds of cure in Staphylococcus aureus bacteremic pneumonia patients correlated with higher haemolytic activity of S. aureus strains, longer time between surgery and bacteremia (> 30 days), longer duration of antibiotic therapy, lower acute physiology and total APACHE II scores, lack of persistent fever for > 72 h and higher levels of antibodies against AT (IgG), ClfA (IgM), FnbpA (IgM) and SdrC (IgM).

Background Ventilator-associated pneumonia caused by Pseudomonas aeruginosa (PA) in hospitalised patients is associated with high mortality. The effectiveness of the bivalent, bispecific mAb MEDI3902 (gremubamab) in preventing PA nosocomial pneumonia was assessed in PA-colonised mechanically ventilated subjects. Methods EVADE (NCT02696902) was a phase 2, randomised, parallel-group, double-blind, placebo-controlled study in Europe, Turkey, Israel, and the USA. Subjects ≥ 18 years old, mechanically ventilated, tracheally colonised with PA, and without new-onset pneumonia, were randomised (1:1:1) to MEDI3902 500, 1500 mg (single intravenous dose), or placebo. The primary efficacy endpoint was the incidence of nosocomial PA pneumonia through 21 days post-dose in MEDI3902 1500 mg versus placebo, determined by an independent adjudication committee. Results Even if the initial sample size was not reached because of low recruitment, 188 subjects were randomised (MEDI3902 500/1500 mg: n  = 16/87; placebo: n  = 85) between 13 April 2016 and 17 October 2019. Out of these, 184 were dosed (MEDI3902 500/1500 mg: n  = 16/85; placebo: n  = 83), comprising the modified intent-to-treat set. Enrolment in the 500 mg arm was discontinued due to pharmacokinetic data demonstrating low MEDI3902 serum concentrations. Subsequently, enrolled subjects were randomised (1:1) to MEDI3902 1500 mg or placebo. PA pneumonia was confirmed in 22.4% ( n  = 19/85) of MEDI3902 1500 mg recipients and in 18.1% ( n  = 15/83) of placebo recipients (relative risk reduction [RRR]: − 23.7%; 80% confidence interval [CI] − 83.8%, 16.8%; p  = 0.49). At 21 days post-1500 mg dose, the mean (standard deviation) serum MEDI3902 concentration was 9.46 (7.91) μg/mL, with 80.6% ( n  = 58/72) subjects achieving concentrations > 1.7 μg/mL, a level associated with improved outcome in animal models. Treatment-emergent adverse event incidence was similar between groups. Conclusions The bivalent, bispecific monoclonal antibody MEDI3902 (gremubamab) did not reduce PA nosocomial pneumonia incidence in PA-colonised mechanically ventilated subjects. Trial registration Registered on Clinicaltrials.gov ( NCT02696902 ) on 11th February 2016 and on EudraCT ( 2015-001706-34 ) on 7th March 2016.