Hyperbaric oxygen therapy augments ciprofloxacin effect against Pseudomonas aeruginosa biofilm infected chronic wounds in a mouse model.

Introduction: Chronic wounds have a compromised microcirculation which leads to restricted gas exchange. The majority of these hypoxic wounds is infested with microorganisms congregating in biofilms which further hinders the antibiotic function. We speculate whether this process can be counteracted by hyperbaric oxygen therapy (HBOT). Methodology: Twenty-eight BALB/c mice with third-degree burns were included in the analyses. Pseudomonas aeruginosa embedded in seaweed alginate beads was injected under the eschar to mimic a biofilm infected wound. Challenged mice were randomized to receive either 4 days with 1 x ciprofloxacin combined with 2 × 90 min HBOT at 2.8 standard atmosphere daily, 1 x ciprofloxacin as monotherapy or saline as placebo. The mice were clinically scored, and wound sizes were estimated by planimetry daily. Euthanasia was performed on day 8. Wounds were surgically removed in toto, homogenized and plated for quantitative bacteriology. Homogenate supernatants were used for cytokine analysis. Results: P. aeruginosa was present in all wounds at euthanasia. A significant lower bacterial load was seen in the HBOT group compared to either the monotherapy ciprofloxacin group (p = 0.0008), or the placebo group (p < 0.0001). IL-1ß level was significantly lower in the HBOT group compared to the placebo group (p = 0.0007). Both treatment groups had higher osteopontin levels than the placebo group (p = 0.002 and p = 0.004). The same pattern was seen in the S100A9 analysis (p = 0.01 and p = 0.008), whereas no differences were detected between the S100A8, the VEGF or the MMP8 levels in the three groups. Conclusion: These findings show that HBOT improves the bactericidal activity of ciprofloxacin against P. aeruginosa wound biofilm in vivo. HBOT in addition to ciprofloxacin also modulates the host response to a less inflammatory phenotype.

Improved Healing of Diabetic Foot Ulcers After High-dose Vitamin D: A Randomized Double-blinded Clinical Trial.

Background. Chronic foot ulcers are a major cause of morbidity in people with diabetes with a lifetime risk of 25%. Treatment is challenging and the recurrence rates of foot ulcers are >50% after 3 years. Vitamin D deficiency is more common in people with diabetes with chronic foot ulcers, compared to both people without diabetes as well as people with diabetes but without foot ulcers. Purpose/aim of study. To assess the efficacy of high-dose compared to low-dose Cholecalciferol vitamin D3 on healing of chronic diabetic foot ulcers. Materials and methods. We included people with diabetes with one or more foot ulcers lasting for more than 6 weeks. Patients were randomly allocated to either a daily oral intake of high-dose (170 µg) or low-dose (20 µg) vitamin D3 (Cholecalciferol). We saw patients in the outpatient clinic after 4, 12, 24, 36, and 48 weeks. At each visit, we measured the ulcer with a specialized camera, and associated software and the area (cm2) was calculated. Patients and assessors were blinded to treatment allocation. We followed all patients for 48 weeks or until wound healing or surgical treatment. Findings/results. We included 48 patients in the analysis (24 in each group), with a total of 64 ulcers. Among them 41 ulcers were followed until healing or 48-week follow-up and 20 ulcers were surgically treated during the study period. Three patients were lost for follow-up. The intention-to-treat analysis showed a significantly higher rate of ulcer healing in the high-dose group with 21 of 30 (70%) healed ulcers compared to 12 of 34 (35%) in the low-dose group (P = .012). Median ulcer reduction at final follow-up was 100% (interquartile range [IQR]: 72-100) in the high-dose group and 57% (IQR: -28 to 100) in the low-dose group. Furthermore, we found a significant effect of high-dose vitamin D on ulcer reduction in the repeated measures analysis of variance. Conclusions. We found high-dose vitamin D3 to be efficient, compared to low-dose vitamin D3, in promoting healing in chronic diabetic foot ulcers.

Potential Advances of Adjunctive Hyperbaric Oxygen Therapy in Infective Endocarditis.

Patients with infective endocarditis (IE) form a heterogeneous group by age, co-morbidities and severity ranging from stable patients to patients with life-threatening complications with need for intensive care. A large proportion need surgical intervention. In-hospital mortality is 15-20%. The concept of using hyperbaric oxygen therapy (HBOT) in other severe bacterial infections has been used for many decades supported by various preclinical and clinical studies. However, the availability and capacity of HBOT may be limited for clinical practice and we still lack well-designed studies documenting clinical efficacy. In the present review we highlight the potential beneficial aspects of adjunctive HBOT in patients with IE. Based on the pathogenesis and pathophysiological conditions of IE, we here summarize some of the important mechanisms and effects by HBOT in relation to infection and inflammation in general. In details, we elaborate on the aspects and impact of HBOT in relation to the host response, tissue hypoxia, biofilm, antibiotics and pathogens. Two preclinical (animal) studies have shown beneficial effect of HBOT in IE, but so far, no clinical study has evaluated the feasibility of HBOT in IE. New therapeutic options in IE are much needed and adjunctive HBOT might be a therapeutic option in certain IE patients to decrease morbidity and mortality and improve the long-term outcome of this severe disease.

Distinct contribution of hyperbaric oxygen therapy to human neutrophil function and antibiotic efficacy against Staphylococcus aureus.

Staphylococcus aureus (SA) causes superficial and severe endovascular infections. The present in vitro study investigates the anti-SA mechanisms of hyperbaric oxygen therapy (HBOT) on direct bacterial killing, antibiotic potentiation, and polymorphonuclear leukocyte (PMN) enhancement. SA was exposed to isolated human PMNs, tobramycin, ciprofloxacin, or benzylpenicillin. HBOT was used as one 90-min session. Bacterial survival was evaluated after 4 h by quantitative bacteriology. PMN functionality as reactive oxygen species (ROS) production was measured by means of dihydrorhodamine 123 analysis. We showed that HBOT exhibits significant direct anti-SA effects. HBOT increased the anti-SA effects of PMNs by 18% after PMA stimulation (p = 0.0004) and by 15% in response to SA (p = 0.36). HBOT showed an additive effect as growth reductions of 26% to sub-MICs of tobramycin (p = 0.0057), 44% to sub-MICs of ciprofloxacin (p = 0.0001), and 26% to sub-MICs of penicillin (p = 0.038). The present in vitro study provides evidence that HBOT has differential mechanisms mediating its anti-SA effects. Our observation supports the clinical possibility for adjunctive HBOT to augment the host immune response and optimize the efficacy of antibiotic treatments.

Hyperbaric oxygen treatment impacts oxidative stress markers in patients with necrotizing soft-tissue infection.

Necrotizing soft-tissue infection (NSTI) is a rare, severe, and fast-progressing bacterial infection associated with a high risk of developing sepsis or septic shock. Increasing evidence indicates that oxidative stress is crucial in the development and progression of sepsis, but its role in NSTI specifically has not been investigated. Some patients with NSTI receive hyperbaric oxygen (HBO2) treatment as the restoration of oxidative stress balance is considered an important mechanism of action, which HBO2 facilitates. However, a gap in knowledge exists regarding the effect of HBO2 treatment on oxidative stress in patients with NSTI. In the present observational study, we aimed to investigate HBO2 treatment effects on known markers of oxidative stress in patients with NSTI. We measured plasma myeloperoxidase (MPO), superoxide dismutase (SOD), heme oxygenase-1 (HO-1) and nitrite+nitrate in 80 patients with NSTI immediately before and after their first HBO2 treatment, and on the following day. We found that HBO2 treatment was associated with a significant increase in MPO and SOD by a median of 3.4 and 8.8 ng/mL, respectively. Moreover, we observed an HBO2 treatment-associated increase in HO-1 in patients presenting with septic shock (n=39) by a median of 301.3 pg/mL. All markers were significantly higher in patients presenting with septic shock compared to patients without shock, and all markers correlated with disease severity. High baseline SOD was associated with 90-day mortality. In conclusion, HBO2 treatment was associated with an increase in MPO and SOD in patients with NSTI, and oxidative stress was more pronounced in patients with septic shock.

Soluble ICAM-1 is modulated by hyperbaric oxygen treatment and correlates with disease severity and mortality in patients with necrotizing soft-tissue infection.

The inflammatory response in patients with necrotizing soft-tissue infection (NSTI) is excessive and often causes collateral damage, thereby worsening disease severity and prognosis. Shedding of endothelial adhesion molecules may be a key regulatory mechanism to modulate the inflammatory response in patients with septic NSTI. Hyperbaric oxygen (HBO2) treatment has demonstrated an effect on adhesion molecules. However, endothelial shedding and its association with NSTI disease severity and prognosis is not fully understood. We hypothesized that shedding of intercellular adhesion molecule-1, and the resulting release of the soluble isoform soluble intercellular adhesion molecule-1 (sICAM-1), is modified by HBO2 treatment, and that sICAM-1 concentrations are associated with severity of disease and mortality in patients with NSTI. We measured sICAM-1 in 80 patients with NSTI immediately before and after first session of HBO2 treatment as well as on the following day. We found an overall sICAM-1 level of 594 ng/mL [interquartile range (IQR) 406-817]. HBO2 significantly (P = 0.01) increased sICAM-1 by a median of 45.1 ng/mL, which remained elevated until the following day; this effect was more pronounced in patients with septic shock. Furthermore, sICAM-1 was significantly correlated with disease severity [simplified acute physiology score II (SAPS II); ρ = 0.24, P = 0.04] and low sICAM-1 was found to be an independent predictor for 90-day mortality in age-sex-SAPS II-adjusted analysis (odds ratio 14.0, 95% CI 1.82-341.4, P = 0.03). These results support the hypothesis that endothelial shedding is an important pathophysiological mechanism in NSTI and suggest that HBO2 treatment may induce immunomodulatory effects that potentially decreases collateral damage and mortality.NEW & NOTEWORTHY HBO2 treatment may be a promising immunomodulatory agent by increasing sICAM-1, thereby lowering risk of collateral damage, especially in the most critically ill patients. sICAM-1 is associated with disease severity in NSTI as emphasized by significant correlations with SAPS II. Low sICAM-1 levels are an independent risk factor of 90-day mortality and appeared to give a good level of diagnostic accuracy, suggesting that sICAM-1 can be used as a prognostic biomarker for NSTI.

Positive effects of neuromuscular shoulder exercises with or without EMG-biofeedback, on pain and function in participants with subacromial pain syndrome - A randomised controlled trial.

BACKGROUND: The aim was to investigate the effect of Electromyography (EMG)-biofeedback guided exercises (BIONEX) on shoulder pain and function in participants with subacromial pain syndrome (SPS). METHODS: Twenty-five women and 24 men (19-67 years), diagnosed with SPS, were randomised to BIONEX or the same exercises without EMG-biofeedback (NEX). Primary outcome was shoulder pain during the past 7 days (Numeric Pain Rating Scale (NPRS)). Secondary outcomes included self-reported (Disability of Arm Shoulder and Hand (DASH), Oxford Shoulder Score (OSS)), and measured shoulder function (surface EMG from upper trapezius, lower trapezius and serratus anterior) in mean and ratios of % of maximum voluntary EMG (%MVE) and onset time (msec), during arm tasks with 0, 1 and 3 kg. RESULTS: There was no group difference (BIONEX versus NEX) in changed shoulder pain (NPRS, mean difference 0.18 (95% CI. -1.56; 1.19)), self-reported or measured shoulder function. Both groups, however, showed significant within-group improvements on self-reported outcomes (NPRS, DASH, OSS), only clinically relevant on NPRS (BIONEX 2.23 (SD 2.47); NEX 2.04 (SD 2.29)). CONCLUSION: BIONEX and NEX were both effective in reducing pain to a clinically relevant level, while EMG-biofeedback did not make a difference. The current neuromuscular shoulder exercise protocol is recommended.

Antimicrobial Tolerance and Metabolic Adaptations in Microbial Biofilms.

Active bacterial metabolism is a prerequisite for optimal activity of many classes of antibiotics. Hence, bacteria have developed strategies to reduce or modulate metabolic pathways to become tolerant. This review describes the tight relationship between metabolism and tolerance in bacterial biofilms, and how physicochemical properties of the microenvironment at the host-pathogen interface (such as oxygen and nutritional content) are key to this relationship. Understanding how metabolic adaptations lead to tolerance brings us to novel approaches to tackle antibiotic-tolerant biofilms. We describe the use of hyperbaric oxygen therapy, metabolism-stimulating metabolites, and alternative strategies to redirect bacterial metabolism towards an antibiotic-susceptible phenotype.

Antibiotic therapy as personalized medicine - general considerations and complicating factors.

The discovery of antibiotic drugs is considered one of the previous century's most important medical discoveries (Medicine's 10 greatest discoveries. New Haven, CT: Yale University Press, 1998: 263). Appropriate use of antibiotics saves millions of lives each year and prevents infectious complications for numerous people. Still, infections kill unacceptable many people around the world, even in developed countries with easy access to most antibiotic drugs. Optimal use of antibiotics is dependent on the identification of primary and secondary focus, and knowledge on which pathogens to expect in a specific infectious syndrome and information on general patterns of regional antibiotic resistance. Furthermore, sampling for microbiological analysis, knowledge of patient immune status and organ functions, travel history, pharmacokinetics and -dynamics of the different antibiotics and possible biofilm formation are among several factors involved in antibiotic therapy of infectious diseases. The present review aims at describing important considerations when using antibacterial antibiotics and to describe how this is becoming substantially more personalized. The parameters relevant in considering the optimal use of antibiotics to treat infections are shown in Fig. 1 - leading to the most relevant antibiotic therapy for that specific patient. To illustrate this subject, the present review's focus will be on challenges with optimal dosing of antibiotics and risks of underdosing. Especially, in cases highly challenging for achieving the aimed antibiotic effect against bacterial infections - this includes augmented renal clearance (ARC) in sepsis, dosing challenges of antibiotics in pregnancy and against biofilm infections.

Development of a novel, robust and cost-efficient process for valorizing dairy waste exemplified by ethanol production.

BACKGROUND: Delactosed whey permeate (DWP) is a side stream of whey processing, which often is discarded as waste, despite of its high residual content of lactose, typically 10-20%. Microbial fermentation is one of the most promising approaches for valorizing nutrient rich industrial waste streams, including those generated by the dairies. Here we present a novel microbial platform specifically designed to generate useful compounds from dairy waste. As a starting point we use Corynebacterium glutamicum, an important workhorse used for production of amino acids and other important compounds, which we have rewired and complemented with genes needed for lactose utilization. To demonstrate the potential of this novel platform we produce ethanol from lactose in DWP. RESULTS: First, we introduced the lacSZ operon from Streptococcus thermophilus, encoding a lactose transporter and a ß-galactosidase, and achieved slow growth on lactose. The strain could metabolize the glucose moiety of lactose, and galactose accumulated in the medium. After complementing with the Leloir pathway (galMKTE) from Lactococcus lactis, co-metabolization of galactose and glucose was accomplished. To further improve the growth and increase the sugar utilization rate, the strain underwent adaptive evolution in lactose minimal medium for 100 generations. The outcome was strain JS95 that grew fast in lactose mineral medium. Nevertheless, JS95 still grew poorly in DWP. The growth and final biomass accumulation were greatly stimulated after supplementation with NH4+, Mn2+, Fe2+ and trace minerals. In only 24 h of cultivation, a high cell density (OD600 of 56.8 ± 1.3) was attained. To demonstrate the usefulness of the platform, we introduced a plasmid expressing pyruvate decarboxylase and alcohol dehydrogenase, and managed to channel the metabolic flux towards ethanol. Under oxygen-deprived conditions, non-growing suspended cells could convert 100 g/L lactose into 46.1 ± 1.4 g/L ethanol in DWP, a yield of 88% of the theoretical. The resting cells could be re-used at least three times, and the ethanol productivities obtained were 0.96 g/L/h, 2.2 g/L/h, and 1.6 g/L/h, respectively. CONCLUSIONS: An efficient process for producing ethanol from DWP, based on C. glutamicum, was demonstrated. The results obtained clearly show a great potential for this newly developed platform for producing value-added chemicals from dairy waste.

Hyperbaric oxygen treatment increases killing of aggregating Pseudomonas aeruginosa isolates from cystic fibrosis patients.

BACKGROUND: Pseudomonas aeruginosa is a major pathogen of the chronic lung infections in cystic fibrosis (CF) patients. These persistent bacterial infections are characterized by bacterial aggregates with biofilm-like properties and are treated with nebulized or intravenous tobramycin in combination with other antibiotics. However, the chronic infections are close to impossible to eradicate due to reasons that are far from fully understood. Recent work has shown that re­oxygenation of hypoxic aggregates by hyperbaric oxygen (O2) treatment (HBOT: 100% O2 at 2.8 bar) will increase killing of aggregating bacteria by antibiotics. This is relevant for treatment of infected CF patients where bacterial aggregates are found in the endobronchial secretions that are depleted of O2 by the metabolism of polymorphonuclear leukocytes (PMNs). The main objective of this study was to investigate the effect of HBOT as an adjuvant to tobramycin treatment of aggregates formed by P. aeruginosa isolates from CF patients. METHODS: The effect was tested using a model with bacterial aggregates embedded in agarose. O2 profiling was used to confirm re­oxygenation of aggregates. RESULTS: We found that HBOT was able to significantly enhance the effect of tobramycin against aggregates of all the P. aeruginosa isolates in vitro. The effect was attributed to increased O2 levels leading to increased growth and thus increased uptake of and killing by tobramycin. CONCLUSIONS: Re­oxygenation may in the future be a clinical possibility as adjuvant to enhance killing by antibiotics in cystic fibrosis lung infections.

Modelling of ciprofloxacin killing enhanced by hyperbaric oxygen treatment in Pseudomonas aeruginosa PAO1 biofilms.

OUTLINE: In chronic lung infections by Pseudomonas aeruginosa (PA) the bacteria thrive in biofilm structures protected from the immune system of the host and from antibiotic treatment. Increasing evidence suggests that the susceptibility of the bacteria to antibiotic treatment can be significantly enhanced by hyperbaric oxygen treatment. The aim of this study is to simulate the effect of ciprofloxacin treatment in a PAO1 biofilm model with aggregates in agarose when combined with hyperbaric oxygen treatment. This is achieved in a reaction-diffusion model that describes the combined effect of ciprofloxacin diffusion, oxygen diffusion and depletion, bacterial growth and killing, and adaptation of the bacteria to ciprofloxacin. In the model, the oxygen diffusion and depletion use a set of parameters derived from experimental results presented in this work. The part of the model describing ciprofloxacin killing uses parameter values from the literature in combination with our estimates (Jacobs, et al., 2016; Grillon, et al., 2016). Micro-respirometry experiments were conducted to determine the oxygen consumption in the P. aeruginosa strain PAO1. The parameters were validated against existing data from an HBOT experiment by Kolpen et al. (2017). The complete oxygen model comprises a reaction-diffusion equation describing the oxygen consumption by using a Michaelis-Menten reaction term. The oxygen model performed well in predicting oxygen concentrations in both time and depth into the biofilm. At 2.8 bar pure oxygen pressure, HBOT increases the penetration depth of oxygen into the biofilm almost by a of factor 4 in agreement with the scaling that follows from the stationary balance between the consumption term and diffusion term. CONCLUSION: In the full reaction-diffusion model we see that hyperbaric oxygen treatment significantly increases the killing by ciprofloxacin in a PAO1 biofilm in alignment with the experimental results from Kolpen et al. (Kolpen, et al., 2017; Kolpen, et al. 2016). The enhanced killing, in turn, lowers the oxygen consumption in the outer layers of the biofilm, and leads to even deeper penetration of oxygen into the biofilm.

Direct evidence of a large Northern European Roman period martial event and postbattle corpse manipulation.

New archaeological excavations at Alken Enge, Jutland, Denmark, have revealed a comprehensive assemblage of disarticulated human remains within a 75-ha wetland area. A minimum of 82 individuals have been uncovered. Based on the distribution, the total population is estimated to be greater than 380 individuals, exclusively male and predominantly adult. The chronological radiocarbon evidence of the human bones indicates that they belong to a single, large event in the early first century AD. The bones show a high frequency of unhealed trauma from sharp-edged weapons, which, together with finds of military equipment, suggests that the find is of martial character. Taphonomic traces indicate that the bones were exposed to animal gnawing for a period of between 6 mo and 1 y before being deposited in the lake. Furthermore, the find situations, including collections of bones, ossa coxae threaded onto a stick, and cuts and scraping marks, provide evidence of the systematic treatment of the human corpses after the time of exposure. The finds are interpreted as the remains of an organized and possibly ritually embedded clearing of a battlefield, including the physical manipulation of the partly skeletonized bones of the deceased fighters and subsequent deposition in the lake. The date places the finds in the context of the Germanic region at the peak of the Roman expansion northward and provides the earliest direct archaeological evidence of large-scale conflict among the Germanic populations and a demonstration of hitherto unrecognized postbattle practices.

The Basic Science of Behavior Change and Its Application to Pediatric Providers.

Pediatric primary care providers (PPCPs) are increasingly expected to know how to assess, diagnose, and treat a wide range of mental health problems in children and adolescents. For many PPCPs, this means learning and performing new practice behaviors that were not taught in their residency training. Typical continuing education approaches to engage PPCPs in new practices have not yielded the desired changes in provider behavior. This article summarizes behavior change principles identified through basic behavior science, adult education, and communication research, and discusses their application to a patient-centered pediatric primary care mental health curriculum.

Hyperbaric Oxygen Sensitizes Anoxic Pseudomonas aeruginosa Biofilm to Ciprofloxacin.

Chronic Pseudomonas aeruginosa lung infection is characterized by the presence of endobronchial antibiotic-tolerant biofilm, which is subject to strong oxygen (O2) depletion due to the activity of surrounding polymorphonuclear leukocytes. The exact mechanisms affecting the antibiotic susceptibility of biofilms remain unclear, but accumulating evidence suggests that the efficacy of several bactericidal antibiotics is enhanced by stimulation of aerobic respiration of pathogens, while lack of O2 increases their tolerance. In fact, the bactericidal effect of several antibiotics depends on active aerobic metabolism activity and the endogenous formation of reactive O2 radicals (ROS). In this study, we aimed to apply hyperbaric oxygen treatment (HBOT) to sensitize anoxic P. aeruginosa agarose biofilms established to mimic situations with intense O2 consumption by the host response in the cystic fibrosis (CF) lung. Application of HBOT resulted in enhanced bactericidal activity of ciprofloxacin at clinically relevant durations and was accompanied by indications of restored aerobic respiration, involvement of endogenous lethal oxidative stress, and increased bacterial growth. The findings highlight that oxygenation by HBOT improves the bactericidal activity of ciprofloxacin on P. aeruginosa biofilm and suggest that bacterial biofilms are sensitized to antibiotics by supplying hyperbaric O2.

Finding the Needle in the Haystack-the Use of Microfluidic Droplet Technology to Identify Vitamin-Secreting Lactic Acid Bacteria.

Efficient screening technologies aim to reduce both the time and the cost required for identifying rare mutants possessing a phenotype of interest in a mutagenized population. In this study, we combined a mild mutagenesis strategy with high-throughput screening based on microfluidic droplet technology to identify Lactococcus lactis variants secreting vitamin B2 (riboflavin). Initially, we used a roseoflavin-resistant mutant of L. lactis strain MG1363, JC017, which secreted low levels of riboflavin. By using fluorescence-activated droplet sorting, several mutants that secreted riboflavin more efficiently than JC017 were readily isolated from the mutagenesis library. The screening was highly efficient, and candidates with as few as 1.6 mutations per million base pairs (Mbp) were isolated. The genetic characterization revealed that riboflavin production was triggered by mutations inhibiting purine biosynthesis, which is surprising since the purine nucleotide GTP is a riboflavin precursor. Purine starvation in the mutants induced overexpression of the riboflavin biosynthesis cluster ribABGH When the purine starvation was relieved by purine supplementation in the growth medium, the outcome was an immediate downregulation of the riboflavin biosynthesis cluster and a reduction in riboflavin production. Finally, by applying the new isolates in milk fermentation, the riboflavin content of milk (0.99 mg/liter) was improved to 2.81 mg/liter, compared with 0.66 mg/liter and 1.51 mg/liter by using the wild-type strain and the original roseoflavin-resistant mutant JC017, respectively. The results obtained demonstrate how powerful classical mutagenesis can be when combined with droplet-based microfluidic screening technology for obtaining microorganisms with useful attributes.IMPORTANCE The food industry prefers to use classical approaches, e.g., random mutagenesis followed by screening, to improve microorganisms used in food production, as the use of recombinant DNA technologies is still not widely accepted. Although modern automated screening platforms are widely accessible, screening remains as a bottleneck in strain development, especially when a mild mutagenesis approach is applied to reduce the chance of accumulating unintended mutations, which may cause unwanted phenotypic changes. Here, we incorporate a droplet-based high-throughput screening method into the strain development process and readily capture L. lactis variants with more efficient vitamin secretion from low-error-rate mutagenesis libraries. This study shows that useful mutants showing strong phenotypes but without extensive mutations can be identified with efficient screening technologies. It is therefore possible to avoid accumulating detrimental mutations while enriching beneficial ones through iterative mutagenesis screening. Due to the low mutation rates, the genetic determinants are also readily identified.

Pseudomonas aeruginosa Aggregate Formation in an Alginate Bead Model System Exhibits In Vivo-Like Characteristics.

Alginate beads represent a simple and highly reproducible in vitro model system for diffusion-limited bacterial growth. In this study, alginate beads were inoculated with Pseudomonas aeruginosa and followed for up to 72 h. Confocal microscopy revealed that P. aeruginosa formed dense clusters similar in size to in vivo aggregates observed ex vivo in cystic fibrosis lungs and chronic wounds. Bacterial aggregates primarily grew in the bead periphery and decreased in size and abundance toward the center of the bead. Microsensor measurements showed that the O2 concentration decreased rapidly and reached anoxia ∼100 µm below the alginate bead surface. This gradient was relieved in beads supplemented with NO3- as an alternative electron acceptor allowing for deeper growth into the beads. A comparison of gene expression profiles between planktonic and alginate-encapsulated P. aeruginosa confirmed that the bacteria experienced hypoxic and anoxic growth conditions. Furthermore, alginate-encapsulated P. aeruginosa exhibited a lower respiration rate than the planktonic counterpart and showed a high tolerance toward antibiotics. The inoculation and growth of P. aeruginosa in alginate beads represent a simple and flexible in vivo-like biofilm model system, wherein bacterial growth exhibits central features of in vivo biofilms. This was observed by the formation of small cell aggregates in a secondary matrix with O2-limited growth, which was alleviated by the addition of NO3- as an alternative electron acceptor, and by reduced respiration rates, as well as an enhanced tolerance to antibiotic treatment.IMPORTANCEPseudomonas aeruginosa has been studied intensively for decades due to its involvement in chronic infections, such as cystic fibrosis and chronic wounds, where it forms biofilms. Much research has been dedicated to biofilm formation on surfaces; however, in chronic infections, most biofilms form small aggregates of cells not attached to a surface, but embedded in host material. In this study, bacteria were encapsulated in small alginate beads and formed aggregates similar to what is observed in chronic bacterial infections. Our findings show that aggregates are exposed to steep oxygen gradients, with zones of oxygen depletion, and that nitrate may serve as an alternative to oxygen, enabling growth in oxygen-depleted zones. This is important, as slow growth under low-oxygen conditions may render the bacteria tolerant toward antibiotics. This model provides an alternative to surface biofilm models and adds to the comprehension that biofilms do not depend on a surface for formation.

Biofilm as a production platform for heterologous production of rhamnolipids by the non-pathogenic strain Pseudomonas putida KT2440.

BACKGROUND: Although a transition toward sustainable production of chemicals is needed, the physiochemical properties of certain biochemicals such as biosurfactants make them challenging to produce in conventional bioreactor systems. Alternative production platforms such as surface-attached biofilm populations could potentially overcome these challenges. Rhamnolipids are a group of biosurfactants highly relevant for industrial applications. However, they are mainly produced by the opportunistic pathogen Pseudomonas aeruginosa using hydrophobic substrates such as plant oils. As the biosynthesis is tightly regulated in P. aeruginosa a heterologous production of rhamnolipids in a safe organism can relive the production from many of these limitations and alternative substrates could be used. RESULTS: In the present study, heterologous production of biosurfactants was investigated using rhamnolipids as the model compound in biofilm encased Pseudomonas putida KT2440. The rhlAB operon from P. aeruginosa was introduced into P. putida to produce mono-rhamnolipids. A synthetic promoter library was used in order to bypass the normal regulation of rhamnolipid synthesis and to provide varying expression levels of the rhlAB operon resulting in different levels of rhamnolipid production. Biosynthesis of rhamnolipids in P. putida decreased bacterial growth rate but stimulated biofilm formation by enhancing cell motility. Continuous rhamnolipid production in a biofilm was achieved using flow cell technology. Quantitative and structural investigations of the produced rhamnolipids were made by ultra performance liquid chromatography combined with high resolution mass spectrometry (HRMS) and tandem HRMS. The predominant rhamnolipid congener produced by the heterologous P. putida biofilm was mono-rhamnolipid with two C10 fatty acids. CONCLUSION: This study shows a successful application of synthetic promoter library in P. putida KT2440 and a heterologous biosynthesis of rhamnolipids in biofilm encased cells without hampering biofilm capabilities. These findings expands the possibilities of cultivation setups and paves the way for employing biofilm flow systems as production platforms for biochemicals, which as a consequence of physiochemical properties are troublesome to produce in conventional fermenter setups, or for production of compounds which are inhibitory or toxic to the production organisms.

Cell Line Derived 5-FU and Irinotecan Drug-Sensitivity Profiles Evaluated in Adjuvant Colon Cancer Trial Data.

PURPOSE: This study evaluates whether gene signatures for chemosensitivity for irinotecan and 5-fluorouracil (5-FU) derived from in vitro grown cancer cell lines can predict clinical sensitivity to these drugs. METHODS: To test if an irinotecan signature and a SN-38 signature could identify patients who benefitted from the addition of irinotecan to 5-FU, we used gene expression profiles based on cell lines and clinical tumor material. These profiles were applied to expression data obtained from pretreatment formalin fixed paraffin embedded (FFPE) tumor tissue from 636 stage III colon cancer patients enrolled in the PETACC-3 prospective randomized clinical trial. A 5-FU profile developed similarly was assessed by comparing the PETACC-3 cohort with a cohort of 359 stage II colon cancer patients who underwent surgery but received no adjuvant therapy. RESULTS: There was no statistically significant association between the irinotecan or SN-38 profiles and benefit from irinotecan. The 5-FU sensitivity profile showed a statistically significant association with relapse free survival (RFS) (hazard ratio (HR) = 0.54 (0.41-0.71), p<1e-05) and overall survival (HR = 0.47 (0.34-0.63), p<1e-06) in the PETACC-3 subpopulation. The effect of the 5-FU profile remained significant in a multivariable Cox Proportional Hazards model, adjusting for several relevant clinicopathological parameters. No statistically significant effect of the 5-FU profile was observed in the untreated cohort of 359 patients (relapse free survival, p = 0.671). CONCLUSION: The irinotecan predictor had no predictive value. The 5-FU predictor was prognostic in stage III patients in PETACC-3 but not in stage II patients with no adjuvant therapy. This suggests a potential predictive ability of the 5-FU sensitivity profile to identify colon cancer patients who may benefit from 5-FU, however, any biomarker predicting benefit for adjuvant 5-FU must be rigorously evaluated in independent cohorts. Given differences between the two study cohorts, the present results should be further validated.