High-Throughput Bacteriophage Testing with Potency Determination: Validation of an Automated Pipetting and Phage Drop-Off Method.

The development of bacteriophages (phages) as active pharmaceutical ingredients for the treatment of patients is on its way and regulatory agencies are calling for reliable methods to assess phage potency. As the number of phage banks is increasing, so is the number of phages that need to be tested to identify therapeutic candidates. Currently, assessment of phage potency on a semi-solid medium to observe plaque-forming units is unavoidable and proves to be labor intensive when considering dozens of phage candidates. Here, we present a method based on automated pipetting and phage drop-off performed by a liquid-handling robot, allowing high-throughput testing and phage potency determination (based on phage titer and efficiency of plaquing). Ten phages were tested, individually and assembled into one cocktail, against 126 Escherichia coli strains. This automated method was compared to the reference one (manual assay) and validated in terms of reproducibility and concordance (ratio of results according to the Bland and Altman method: 0.99; Lin's concordance correlation coefficient: 0.86). We found that coefficients of variation were lower with automated pipetting (mean CV: 13.3% vs. 24.5%). Beyond speeding up the process of phage screening, this method could be used to standardize phage potency evaluation.

Statistical approach for radioactivity detection: A brief review.

Radioactivity detection is a major research and development priority for many practical applications. Amongst the various technical challenges in this field is the need to carry out accurate low-level radioactivity measurements in the presence of a large fluctuations in the natural radiation background, while reducing the false alarm rates. The task becomes even more harder with high detection limits under low signal-to-background ratios. A detection method based on the statistical inference, following either a frequentist or a Bayesian paradigm, adopted to overcome these challenges as well as to ensure a reliable and accurate diagnosis with a competitive tradeoff between sensitivity, specificity and response time. With this respect, several research studies, addressing a range of applications from decommissioning and dismantling to homeland security, have been proposed. Our main goal in this paper is to present a succinct survey of these studies based on a frequentist and Bayesian approaches used to decision-making, uncertainty and risk evaluation, in the context of radioactive detection. In this prospect, a theoretical background of statistical frequentist and Bayesian inferences was presented. Then, a comparative study of both approaches was performed to determine the optimal approach in regards to accuracy and pros/cons. A case of study for low-level radioactivity detection in nuclear decommissioning operations was provided to validate the optimal approach. Results proved the efficiency and usefulness of Bayesian approach against frequentist one with respect to the most challenging scenarios in radiation detection applications.

Combination of in vivo phage therapy data with in silico model highlights key parameters for pneumonia treatment efficacy.

The clinical (re)development of bacteriophage (phage) therapy to treat antibiotic-resistant infections faces the challenge of understanding the dynamics of phage-bacteria interactions in the in vivo context. Here, we develop a general strategy coupling in vitro and in vivo experiments with a mathematical model to characterize the interplay between phage and bacteria during pneumonia induced by a pathogenic strain of Escherichia coli. The model allows the estimation of several key parameters for phage therapeutic efficacy. In particular, it quantifies the impact of dose and route of phage administration as well as the synergism of phage and the innate immune response on bacterial clearance. Simulations predict a limited impact of the intrinsic phage characteristics in agreement with the current semi-empirical choices of phages for compassionate treatments. Model-based approaches will foster the deployment of future phage-therapy clinical trials.

A Case of Phage Therapy against Pandrug-Resistant Achromobacter xylosoxidans in a 12-Year-Old Lung-Transplanted Cystic Fibrosis Patient.

Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains.

When a Ventilator Takes Autonomous Decisions without Seeking Approbation nor Warning Clinicians: A Case Series.

BACKGROUND: Complexity and functions of automated medical devices used to support life (eg, ventilators, dialysis machines, monitors, insulin pump with continuous blood glucose monitoring system, etc.) increase over time. Until recently, devices were partially automated by very simple feedback loops, with no or few software dependence (such as the simplest home thermostat). For the last two decades, devices have been increasingly driven by complex algorithms devoted to improve patient's treatment and monitoring as well as users experience. METHODS: We report the unexpected and inappropriate operation of two recent ventilators, associated to potential harmful consequences. We provide both a description of the clinical situations (five ICU patients, archetypal situations) and a test bench analysis. RESULTS: While set in volume mode, these ventilators activated an algorithm dedicated to limit airway pressure when an increase in airway resistance occurred. In such situations, a pressure-like mode was activated (with decelerating inspiratory flow and set pressure, with target of volume). The main consequences observed were that the tidal volume was no longer guaranteed or delivered and that the pressure limitation operated by the algorithm prevented the airway pressure from reaching the high-pressure alarm threshold. CONCLUSION: This led to the silent takeover of commands by the ventilator without clinicians or nurses being aware of it and without any warnings or alarms emission adapted to the severity of the event. Generally speaking, such an algorithm questions the place of automation and its limit when users are not aware of its presence as well as the need for regulation and additional tests before its implementation. Intensivists and respiratory care specialists should remain vigilant regarding the risk of rare but critical events related to unexpected functioning or insufficiently tested equipment during the pre-clinical development phases. They should not neglect misunderstood critical events without having performed sufficient investigations.

Detection and quantification of copper in scrap metal by linac-based neutron activation analysis.

This paper presents the investigation carried out by CEA List and ArcelorMittal R&D in order to assess the potential of linac-based neutron activation analysis to detect and quantify copper in scrap metal. Performances are evaluated using MCNP6 and then validated experimentally using a 6 MeV linac coupled with heavy water. It is shown that (γ, n) reaction cross-sections for deuterium are likely to be undervalued in ENDF/B-VII and suggested that photoneutron production algorithms in Monte Carlo codes should be reexamined.

[Antibacterial applications of bacteriophages]. / Les applications antibactériennes des bactériophages.

In the 1917 article in which Félix d'Hérelle describes his first observations and proposes the name of bacteriophage, he also reports the first use of these viruses to treat bacterial infections, thus giving birth to phage therapy. Soon after antibiotics supplanted bacteriophages. Today, bacteria resistant to multiple antibiotics become a growing public health issue worldwide. This situation has revived research aiming at developing the antibacterial activity of bacteriophages to treat patients as well as diseases in animals and plants. In fact, the areas of applications of bacteriophages as antibacterial are widening as current solutions of chemical nature are questioned. This review summarizes the basic principles of therapeutic applications of bacteriophages and presents recent data in areas where commercial exploitation is occurring or about to emerge.

[A century of research on bacteriophages]. / Un siècle de recherche sur les bactériophages.

Bacteriophages have a prominent place in the living world. They participate to our understanding of the living world through three main aspects : (i) the dissection of the most intimist aspects of viral infection molecular mechanisms (molecular biology), (ii) the description and functioning mechanisms of ecosystems (ecology), and (iii) the adaptive dynamics of integrated viral and host-cell populations (evolution). This review looks back at the genesis of these fundamental findings and draws a picture of the most active fields of current research.

Hemolysis and Plasma Free Hemoglobin During Extracorporeal Membrane Oxygenation Support: From Clinical Implications to Laboratory Details.

Venovenous and venoarterial extracorporeal membrane oxygenation (ECMO) are lifesaving supports that are more and more frequently used in critically ill patients. Despite of major technological improvements observed during the last 20 years, ECMO-associated hemolysis is still a complication that may arise during such therapy. Hemolysis severity, directly appreciated by plasma free hemoglobin concentration, may be present with various intensity, from a nonalarming and tolerable hemolysis to a highly toxic one. Here, we propose a review dedicated to extracorporeal membrane oxygenation (ECMO)-associated hemolysis, with a particular emphasis on pathophysiology, prevalence, and clinical consequences of such complication. We also focus on laboratory assessment of hemolysis and on the limits that have to be known by clinicians to prevent and manage hemolytic events.

Phage Therapy of Pneumonia Is Not Associated with an Overstimulation of the Inflammatory Response Compared to Antibiotic Treatment in Mice.

Supported by years of clinical use in some countries and more recently by literature on experimental models, as well as its compassionate use in Europe and in the United States, bacteriophage (phage) therapy is providing a solution for difficult-to-treat bacterial infections. However, studies of the impact of such treatments on the host remain scarce. Murine acute pneumonia initiated by intranasal instillation of two pathogenic strains of Escherichia coli (536 and LM33) was treated by two specific bacteriophages (536_P1 and LM33_P1; intranasal) or antibiotics (ceftriaxone, cefoxitin, or imipenem-cilastatin; intraperitoneal). Healthy mice also received phages alone. The severity of pulmonary edema, acute inflammatory cytokine concentration (blood and lung homogenates), complete blood counts, and bacterial and bacteriophage counts were determined at early (≤12 h) and late (≥20 h) time points. The efficacy of bacteriophage to decrease bacterial load was faster than with antibiotics, but the two displayed similar endpoints. Bacteriophage treatment was not associated with overinflammation but in contrast tended to lower inflammation and provided a faster correction of blood cell count abnormalities than did antibiotics. In the absence of bacterial infection, bacteriophage 536_P1 promoted a weak increase in the production of antiviral cytokines (gamma interferon [IFN-γ] and interleukin-12 [IL-12]) and chemokines in the lungs but not in the blood. However, such variations were no longer observed when bacteriophage 536_P1 was administered to treat infected animals. The rapid lysis of bacteria by bacteriophages in vivo does not increase the innate inflammatory response compared to that with antibiotic treatment.

New Bacteriophages against Emerging Lineages ST23 and ST258 of Klebsiella pneumoniae and Efficacy Assessment in Galleria mellonella Larvae.

Klebsiella pneumoniae is a bacterial pathogen of high public health importance. Its polysaccharide capsule is highly variable but only a few capsular types are associated with emerging pathogenic sublineages. The aim of this work is to isolate and characterize new lytic bacteriophages and assess their potential to control infections by the ST23 and ST258 K. pneumoniae sublineages using a Galleria mellonella larvae model. Three selected bacteriophages, targeting lineages ST258 (bacteriophages vB_KpnP_KL106-ULIP47 and vB_KpnP_KL106-ULIP54) and ST23 (bacteriophage vB_KpnP_K1-ULIP33), display specificity for capsular types KL106 and K1, respectively. These podoviruses belong to the Autographivirinae subfamily and their genomes are devoid of lysogeny or toxin-associated genes. In a G. mellonella larvae model, a mortality rate of 70% was observed upon infection by K. pneumoniae ST258 and ST23. This number was reduced to 20% upon treatment with bacteriophages at a multiplicity of infection of 10. This work increases the number of characterized bacteriophages infecting K. pneumoniae and provides information regarding genome sequence and efficacy during preclinical phage therapy against two prominent sublineages of this bacterial species.

Hypervirulent Klebsiella pneumoniae, a 5-year study in a French ICU.

PURPOSE: Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a leading cause of severe community-acquired pneumonia, liver abscess and disseminated infection in the Far East. Data regarding the incidence, clinical features and microbiological characteristics related to hvKp infections in the Western world are scarce. METHODOLOGY: The incidence, clinical features and microbiological characteristics of hvKp infections were investigated through a 5-year survey conducted in a single French intensive care unit. K. pneumoniae strains were screened for hypermucoviscosity based on a string test. Multilocus sequence typing and multiplex PCR analysis targeting virulence genes were performed on string test-positive strains. RESULTS: Over a 53-month period, a total of 59 infections due to K. pneumoniae were identified including 26 community-onset infections. Twelve hvKp infections were documented, accounting for 46.1 % of community-acquired K. pneumoniae. Community-acquired pneumonia (n=6), aspiration pneumonia (n=4) and liver abscess (n=2) represented initial sites and mode of infection. Compared to non-hvKp infections, patients with hvKp infections displayed higher rates of multi-organ failure (83.3 % vs 35.7 %; P=0.04), but mortality rates were not different (50 % vs 35 %; P=0.71). Strains K1/ST23 (n=5) and K2/ST86 (n=5) predominated. All hvKp strains displayed wild-type susceptibility. CONCLUSION: hvKp represent a potentially underestimated cause of fatal infections in the Western world.

In Vivo Bacteriophage Biodistribution.

At the dawn of the renaissance of bacteriophage therapy, the full acceptation of bacteriophages as anti-bacterial agents requires the determination of their basic pharmacokinetic parameters. Such data, known for all conventional drugs used in human and veterinary medicine, allow optimizing dose regimens, efficacy, and help to limit toxicity. Here, we describe basic methods to experimentally obtain pharmacokinetic data and give also examples of data calculation to determine key parameters related to the biodistribution and elimination of bacteriophages in vivo.

[Phage therapy: a realistic weapon against multidrug resistant bacteria]. / La phagothérapie - Une arme crédible face à l'antibiorésistance.

The continuous increase in antibiotic resistance among bacteria in infectious diseases associated with the lack of new antibiotics able to circumvent them are urging physicians, researchers and politicians to look for others options for treatments. Among those, phage therapy (use of natural viruses that infect bacteria, called bacteriophages) is one of the most promising approaches. In this review, we first focus on the problematic raised by multidrug resistant bacteria before addressing the main biological characteristics of bacteriophages, as well as the credibility and the relevance of phage therapy. We then introduce human applications, their potentials and limits.

The Lysis of Pathogenic Escherichia coli by Bacteriophages Releases Less Endotoxin Than by ß-Lactams.

BACKGROUND.: Other than numerous experimental data assessing phage therapy efficacy, questions regarding safety of this approach are not sufficiently addressed. In particular, as phages can kill bacterial cells within <10 minutes, the associated endotoxin release (ER) in severe infections caused by gram-negative bacteria could be a matter of concern. METHODS.: Two therapeutic virulent phages and 4 reference antibiotics were studied in vitro for their ability to kill 2 pathogenic strains of Escherichia coli and generate an ER. The early interaction (first 3 hours) between these actors was assessed over time by studying the instantaneous cell viability, the colony-forming unit count, the concentration of free endotoxin released, and the cell morphology under light microscope. RESULTS.: While ß-lactams have a relatively slow effect, both tested phages, as well as amikacin, were able to rapidly abolish the bacterial growth. Even when considering the fastest phage (cell lysis in 9 minutes), the concentrations of phage-induced ER never reached the highest values, which were recorded with antibiotic treatments. Cumulative concentrations of endotoxin over time in phage-treated conditions were lower than those observed with ß-lactams and close to those observed with amikacin. Whereas ß-lactams were responsible for strong cell morphology changes (spheroplast with imipenem, filamentous cells with cefoxitin and ceftriaxone), amikacin and phages did not modify cell shape but produced intracellular inclusion bodies. CONCLUSIONS.: This work provides important and comforting data regarding the safety of phage therapy. Therapeutically relevant phages, with their low endotoxin release profile and fast bactericidal effect, are not inferior to ß-lactams.

Bacteriophage LM33_P1, a fast-acting weapon against the pandemic ST131-O25b:H4 Escherichia coli clonal complex.

OBJECTIVES: Amongst the highly diverse Escherichia coli population, the ST131-O25b:H4 clonal complex is particularly worrisome as it is associated with a high level of antibiotic resistance. The lack of new antibiotics, the worldwide continuous increase of infections caused by MDR bacteria and the need for narrow-spectrum antimicrobial agents have revived interest in phage therapy. In this article, we describe a virulent bacteriophage, LM33_P1, which specifically infects O25b strains, and provide data related to its therapeutic potential. METHODS: A large panel of E. coli strains (n = 283) was used to assess both the specificity and the activity of bacteriophage LM33_P1. Immunology, biochemistry and genetics-based methods confirmed this specificity. Virology methods and sequencing were used to characterize this bacteriophage in vitro, while three relevant mouse models were employed to show its in vivo efficacy. RESULTS: Bacteriophage LM33_P1 exclusively infects O25b E. coli strains with a 70% coverage on sequence types associated with high antibiotic resistance (ST131 and ST69). This specificity is due to an interaction with the LPS mediated by an original tail fibre. LM33_P1 also has exceptional intrinsic properties with a high adsorption constant and produces over 300 virions per cell in <10 min. Using animal pneumonia, septicaemia and urinary tract infection models, we showed the in vivo efficacy of LM33_P1 to reduce the bacterial load in several organs. CONCLUSIONS: Bacteriophage LM33_P1 represents the first weapon that specifically and quickly kills O25b E. coli strains. Therapeutic approaches derived from this bacteriophage could be developed to stop or slow down the spread of the ST131-O25b:H4 drug-resistant clonal complex in humans.

Small Intestine Early Innate Immunity Response during Intestinal Colonization by Escherichia coli Depends on Its Extra-Intestinal Virulence Status.

Uropathogenic Escherichia coli (UPEC) strains live as commensals in the digestive tract of the host, but they can also initiate urinary tract infections. The aim of this work was to determine how a host detects the presence of a new UPEC strain in the digestive tract. Mice were orally challenged with UPEC strains 536 and CFT073, non-pathogenic strain K12 MG1655, and ΔPAI-536, an isogenic mutant of strain 536 lacking all 7 pathogenicity islands whose virulence is drastically attenuated. Intestinal colonization was measured, and cytokine expression was determined in various organs recovered from mice after oral challenge. UPEC strain 536 efficiently colonized the mouse digestive tract, and prior Enterobacteriaceae colonization was found to impact strain 536 colonization efficiency. An innate immune response, detected as the production of TNFα, IL-6 and IL-10 cytokines, was activated in the ileum 48 hours after oral challenge with strain 536, and returned to baseline within 8 days, without a drop in fecal pathogen load. Although inflammation was detected in the ileum, histology was normal at the time of cytokine peak. Comparison of cytokine secretion 48h after oral gavage with E. coli strain 536, CFT073, MG1655 or ΔPAI-536 showed that inflammation was more pronounced with UPECs than with non-pathogenic or attenuated strains. Pathogenicity islands also seemed to be involved in host detection, as IL-6 intestinal secretion was increased after administration of E. coli strain 536, but not after administration of ΔPAI-536. In conclusion, UPEC colonization of the mouse digestive tract activates acute phase inflammatory cytokine secretion but does not trigger any pathological changes, illustrating the opportunistic nature of UPECs. This digestive tract colonization model will be useful for studying the factors controlling the switch from commensalism to pathogenicity.