Should standardized susceptibility testing for microbial biofilms be introduced in clinical practice?

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Comparison of UVB and UVC irradiation disinfection efficacies on Pseudomonas Aeruginosa biofilm

Disinfection routines are important in all clinical applications. The uprising problem of antibiotic resistance has driven major research efforts towards alternative disinfection approaches, involving light-based solutions. Pseudomonasaeruginosa (P. aeruginosa) is a common bacterium that can cause skin, soft tissue, lungs, kidney and urinary tract infections. Moreover, it can be found on and in medical equipment causing often cross infections in hospitals. The objective of this study was to test the efficiency, of two different light-based disinfection treatments, namely UVB and UVC irradiation, on P. aeruginosa biofilms at different growth stages. In our experiments a new type of UV light emitting diodes (LEDs) were used to deliver UV irradiation on the biofilms, in the UVB (296nm) and UVC (266nm) region. The killing rate was studied as a function of dose for 24h grown biofilms. The dose was ramped from 72J/m2 to 10000J/m2. It was shown that UVB irradiation was more effective than UVC irradiation in inactivating P. aeruginosa biofilms. No colony forming units (CFU) were observed for the UVB treated biofilms when the dose was 10000 J/m2 (CFU in control sample: 7.5 x 104). UVB irradiation at a dose of 20000J/m2 on mature biofilms (72h grown) resulted in a 3.9 log killing efficacy. The fact that the wavelength of 296nm exists in daylight and has such disinfection ability on biofilms gives new perspectives for applications within disinfection at hospitals

Nitrous oxide production in sputum from cystic fibrosis patients with chronic Pseudomonas aeruginosa lung infection

Chronic lung infection by Pseudomonas aeruginosa is the major severe complication in cystic fibrosis (CF) patients, where P. aeruginosa persists and grows in biofilms in the endobronchial mucus under hypoxic conditions. Numerous polymorphonuclear leukocytes (PMNs) surround the biofilms and create local anoxia by consuming the majority of O2 for production of reactive oxygen species (ROS). We hypothesized that P. aeruginosa acquires energy for growth in anaerobic endobronchial mucus by denitrification, which can be demonstrated by production of nitrous oxide (N2O), an intermediate in the denitrification pathway. We measured N2O and O2 with electrochemical microsensors in 8 freshly expectorated sputum samples from 7 CF patients with chronic P. aeruginosa infection. The concentrations of NO 3- and NO2- in sputum were estimated by the Griess reagent. We found a maximum median concentration of 41.8 μM N2O (range 1.4-157.9 μM N2O). The concentration of N2O in the sputum was higher below the oxygenated layers. In 4 samples the N2O concentration increased during the initial 6 h of measurements before decreasing for approximately 6 h. Concomitantly, the concentration of NO3- decreased in sputum during 24 hours of incubation. We demonstrate for the first time production of N2O in clinical material from infected human airways indicating pathogenic metabolism based on denitrification. Therefore, P. aeruginosa may acquire energy for growth by denitrification in anoxic endobronchial mucus in CF patients. Such ability for anaerobic growth may be a hitherto ignored key aspect of chronic P. aeruginosa infections that can inform new strategies for treatment and prevention. © 2014 Kolpen et al

Physiological levels of nitrate support anoxic growth by denitrification of Pseudomonas aeruginosa at growth rates reported in cystic fibrosis lungs and sputum

© 2014 Line, Alhede, Kolpen, Kuhl, Ciofu, Bjarnsholt, Moser, Toyofuku, Nomura, H0i'by and Jensen. Chronic Pseudomonas aeruginosa lung infection is the most severe complication in patients with cystic fibrosis (CF). The infection is characterised by the formation of biofilm surrounded by numerous polymorphonuclear leukocytes (PMNs) and strong O2 depletion in the endobronchial mucus. We have reported that O2 is mainly consumed by the activated PMNs, while O2 consumption by aerobic respiration is diminutive and nitrous oxide (N2O) is produced in infected CF sputum. This suggests that the reported growth rates ofP. aeruginosa in lungs and sputum may result from anaerobic respiration using denitrification. The growth rate of P. aeruginosa achieved by denitrification at physiological levels (~400 μM) of nitrate (NO3-) is however, not known. Therefore, we have measured growth rates of anoxic cultures of PAO1 and clinical isolates (n = 12) in LB media supplemented with NO3- and found a significant increase of growth when supplementing PAO1 and clinical isolates with > 150 μM NO3- and 100 μM NO3-, respectively. An essential contribution to growth by denitrification was demonstrated by the inability to establish a significantly increased growth rate by a denitrification deficient ΔnirS-N mutant at <1 mM of NO3-. Activation of denitrification could be achieved by supplementation with as little as 62.5 μM of NO3- according to the significant production of N2O by the nitrous oxide reductase deficient ΔnosZ mutant. Studies of the promoter activity, gene transcripts and enzyme activity of the four N-oxide reductases in PAO1 (Nar, Nir, Nor, Nos) further verified the engagement of denitrification, showing a transient increase in activation and expression and rapid consumption of NO3- followed by a transient increase of NO2-. Growth rates obtained by denitrification in this study were comparable to our reported growth rates in the majority of P. aeruginosa cells in CF lungs and sputum. Thus, we have demonstrated that denitrification is required for P. aeruginosa growth in infected endobronchial CF mucus

Minimum information guideline for spectrophotometric and fluorometric methods to assess biofilm formation in microplates

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bioflm.2019.100010.The lack of reproducibility of published studies is one of the major issues facing the scientific community, and the field of biofilm microbiology has been no exception. One effective strategy against this multifaceted problem is the use of minimum information guidelines. This strategy provides a guide for authors and reviewers on the necessary information that a manuscript should include for the experiments in a study to be clearly interpreted and independently reproduced. As a result of several discussions between international groups working in the area of biofilms, we present a guideline for the spectrophotometric and fluorometric assessment of biofilm formation in microplates. This guideline has been divided into 5 main sections, each presenting a comprehensive set of recommendations. The intention of the minimum information guideline is to improve the quality of scientific communication that will augment interlaboratory reproducibility in biofilm microplate assays.This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska – Curie grant agreement No 722467, as part of the Print-Aid consortium. The information and views set out in this article are those of the authors and do not necessarily reflect the official opinion of the European Union. Neither the European Union institutions and bodies nor any person acting on their behalf may be held responsible for the use which may be made of the information contained therein. This work received additional financial support by: project UID/EQU/00511/2019 - Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE funded by national funds through FCT/MCTES (PIDDAC); Project “LEPABE-2-ECO-INNOVATION” – NORTE-01-0145-FEDER-000005, funded by Norte Portugal Regional Operational Programme (NORTE 2020), under PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).info:eu-repo/semantics/publishedVersio

Phenotypes of Non-Attached Pseudomonas aeruginosa Aggregates Resemble Surface Attached Biofilm

For a chronic infection to be established, bacteria must be able to cope with hostile conditions such as low iron levels, oxidative stress, and clearance by the host defense, as well as antibiotic treatment. It is generally accepted that biofilm formation facilitates tolerance to these adverse conditions. However, microscopic investigations of samples isolated from sites of chronic infections seem to suggest that some bacteria do not need to be attached to surfaces in order to establish chronic infections. In this study we employed scanning electron microscopy, confocal laser scanning microscopy, RT-PCR as well as traditional culturing techniques to study the properties of Pseudomonas aeruginosa aggregates. We found that non-attached aggregates from stationary-phase cultures have comparable growth rates to surface attached biofilms. The growth rate estimations indicated that, independently of age, both aggregates and flow-cell biofilm had the same slow growth rate as a stationary phase shaking cultures. Internal structures of the aggregates matrix components and their capacity to survive otherwise lethal treatments with antibiotics (referred to as tolerance) and resistance to phagocytes were also found to be strikingly similar to flow-cell biofilms. Our data indicate that the tolerance of both biofilms and non-attached aggregates towards antibiotics is reversible by physical disruption. We provide evidence that the antibiotic tolerance is likely to be dependent on both the physiological states of the aggregates and particular matrix components. Bacterial surface-attachment and subsequent biofilm formation are considered hallmarks of the capacity of microbes to cause persistent infections. We have observed non-attached aggregates in the lungs of cystic fibrosis patients; otitis media; soft tissue fillers and non-healing wounds, and we propose that aggregated cells exhibit enhanced survival in the hostile host environment, compared with non-aggregated bacterial populations

In silico analyses of metagenomes from human atherosclerotic plaque samples

Background Through several observational and mechanistic studies, microbial infection is known to promote cardiovascular disease. Direct infection of the vessel wall, along with the cardiovascular risk factors, is hypothesized to play a key role in the atherogenesis by promoting an inflammatory response leading to endothelial dysfunction and generating a proatherogenic and prothrombotic environment ultimately leading to clinical manifestations of cardiovascular disease, e.g., acute myocardial infarction or stroke. There are many reports of microbial DNA isolation and even a few studies of viable microbes isolated from human atherosclerotic vessels. However, high-resolution investigation of microbial infectious agents from human vessels that may contribute to atherosclerosis is very limited. In spite of the progress in recent sequencing technologies, analyzing host-associated metagenomes remain a challenge. Results To investigate microbiome diversity within human atherosclerotic tissue samples, we employed high-throughput metagenomic analysis on: (1) atherosclerotic plaques obtained from a group of patients who underwent endarterectomy due to recent transient cerebral ischemia or stroke. (2) Presumed stabile atherosclerotic plaques obtained from autopsy from a control group of patients who all died from causes not related to cardiovascular disease. Our data provides evidence that suggest a wide range of microbial agents in atherosclerotic plaques, and an intriguing new observation that shows these microbiota displayed differences between symptomatic and asymptomatic plaques as judged from the taxonomic profiles in these two groups of patients. Additionally, functional annotations reveal significant differences in basic metabolic and disease pathway signatures between these groups. Conclusions We demonstrate the feasibility of novel high-resolution techniques aimed at identification and characterization of microbial genomes in human atherosclerotic tissue samples. Our analysis suggests that distinct groups of microbial agents might play different roles during the development of atherosclerotic plaques. These findings may serve as a reference point for future studies in this area of research