The importance of understanding the infectious microenvironment.

Standard doses of antibiotics do not efficiently treat chronic infections of the soft tissue and bone. In this Personal View, we advocate for improving treatment of these infections by taking the infectious microenvironment into account. The infectious microenvironment can cause sensitive bacteria to lose their susceptibility to antibiotics that are effective in standard laboratory susceptibility testing. We propose that bacteria behave substantially different in standard laboratory conditions than they do in actual infections. The infectious microenvironment could impose changes in growth and metabolic activity that result in increased protection against antibiotics. Therefore, we advocate that improved antibiotic treatment of chronic infection is achievable when antibiotics are recommended on the basis of susceptibility testing in relevant in vitro conditions that resemble actual infectious microenvironments. We recommend establishing knowledge of the relevant conditions of the chemical and physical composition of the infectious microenvironment. Recent advances in RNA sequencing, metabolomics, and microscopy have made it possible for the characterisation of the microenvironment of infections and to validate the clinical relevance of in vitro conditions to actual infections.

Delayed neutrophil recruitment allows nascent Staphylococcus aureus biofilm formation and immune evasion.

Biofilms that form on implanted medical devices cause recalcitrant infections. The early events enabling contaminating bacteria to evade immune clearance, before a mature biofilm is established, are poorly understood. Live imaging in vitro demonstrated that Staphylococcus aureus sparsely inoculated on an abiotic surface can go undiscovered by human neutrophils, grow, and form aggregates. Small (~50 µm2) aggregates of attached bacteria resisted killing by human neutrophils, resulting in neutrophil lysis and bacterial persistence. In vivo, neutrophil recruitment to a peritoneal implant was spatially heterogenous, with some bacterial aggregates remaining undiscovered by neutrophils after 24 h. Intravital imaging in mouse skin revealed that attached S. aureus aggregates grew and remained undiscovered by neutrophils for up to 3 h. These results suggest a model in which delayed recruitment of neutrophils to an abiotic implant presents a critical window in which bacteria establish a nascent biofilm and acquire tolerance to neutrophil killing.

Oxygen Restriction Generates Difficult-to-Culture P. aeruginosa.

Induction of a non-culturable state has been demonstrated for many bacteria, e.g., Escherichia coli and various Vibrio spp. In a clinical perspective, the lack of growth due to these non-culturable bacteria can have major consequences for the treatment of patients. Here, we show how anoxic conditioning (restriction of molecular oxygen, O2) generates difficult-to-culture (DTC) bacteria during biofilm growth. A significant subpopulation of Pseudomonas aeruginosa entered a DTC state after anoxic conditioning, ranging from 5 to 90% of the total culturable population, in both planktonic and biofilm models. Anoxic conditioning also generated DTC subpopulations of Staphylococcus aureus and Staphylococcus epidermidis (89 and 42% of the total culturable population, respectively). Growth of the DTC populations were achieved by substituting O2 with 10 mM NO3 - as an alternative electron acceptor for anaerobic respiration or, in the case of P. aeruginosa, by adding sodium pyruvate or catalase as scavengers against reactive oxygen species (ROS) during aerobic respiration. An increase in normoxic plating due to addition of catalase suggests the molecule hydrogen peroxide as a possible mechanism for induction of DTC P. aeruginosa. Anoxic conditioning also generated a true viable but non-culturable (VBNC) population of P. aeruginosa that was not resurrected by substituting O2 with NO3 - during anaerobic respiration. These results demonstrate that habituation to an anoxic micro-environment could complicate diagnostic culturing of bacteria, especially in the case of chronic infections where oxygen is restricted due to the host immune response.

Adjunctive dabigatran therapy improves outcome of experimental left-sided Staphylococcus aureus endocarditis.

BACKGROUND: Staphylococcus aureus is the most frequent and fatal cause of left-sided infective endocarditis (IE). New treatment strategies are needed to improve the outcome. S. aureus coagulase promotes clot and fibrin formation. We hypothesized that dabigatran, could reduce valve vegetations and inflammation in S. aureus IE. METHODS: We used a rat model of severe aortic valve S. aureus IE. All infected animals were randomized to receive adjunctive dabigatran (10 mg/kg b.i.d., n = 12) or saline (controls, n = 11) in combination with gentamicin. Valve vegetation size, bacterial load, cytokine, cell integrins expression and peripheral platelets and neutrophils were assessed 3 days post-infection. RESULTS: Adjunctive dabigatran treatment significantly reduced valve vegetation size compared to controls (p< 0.0001). A significant reduction of the bacterial load in aortic valves was seen in dabigatran group compared to controls (p = 0.02), as well as expression of key pro-inflammatory markers keratinocyte-derived chemokine, IL-6, ICAM-1, TIMP-1, L-selectin (p< 0.04). Moreover, the dabigatran group had a 2.5-fold increase of circulating platelets compared to controls and a higher expression of functional and activated platelets (CD62p+) unbound to neutrophils. CONCLUSION: Adjunctive dabigatran reduced the vegetation size, bacterial load, and inflammation in experimental S. aureus IE.

Implants induce a new niche for microbiomes.

Although much work is being done to develop new treatments, research and knowledge regarding factors underlying implant-related microbial colonization leading to infection are less comprehensive. Presence of microorganisms in and around implants clinically characterized as uninfected remains unknown. The objective of this study was to detect and identify bacteria and fungi on implants from various groups of patients with no prior indications of implant related infections. Patient samples (implants and tissue) were collected from five different hospitals in the Capital region of Denmark. By in-depth microbiological detection methods, we examined the prevalence of bacteria and fungi on 106 clinically uninfected implants from four patient groups (aseptic loosening, healed fractures, craniofacial complications and recently deceased). Of 106 clinically uninfected implants and 39 negative controls investigated, 66% were colonized by bacteria and 40% were colonized by fungi (p < 0.0001 compared to negative controls). A large number of microbes were found to colonize the implants, however, the most prevalent microbes present were not common aetiological agents of implant infections. The findings indicate that implants provide a distinct niche for microbial colonization. These data have broad implications for medical implant recipients, as well as for supporting the idea that the presence of foreign objects in the body alters the human microbiome by providing new colonization niches.

Extracellular hydrogen peroxide measurements using a flow injection system in combination with microdialysis probes - Potential and challenges.

There is a strong need for techniques that can quantify the important reactive oxygen species hydrogen peroxide (H2O2) in complex media and in vivo. We combined chemiluminescence-based H2O2 measurements on a commercially available flow injection analysis (FIA) system with sampling of the analyte using microdialysis probes (MDPs), typically used for measurements in tissue. This allows minimally invasive, quantitative measurements of extracellular H2O2 concentration and dynamics utilizing the chemiluminescent reaction of H2O2 with acridinium ester. By coupling MDPs to the FIA system, measurements are no longer limited to filtered, liquid samples with low viscosity, as sampling via a MDP is based on a dynamic exchange through a permeable membrane with a specific cut-off. This allows continuous monitoring of dynamic changes in H2O2 concentrations, alleviates potential pH effects on the measurements, and allows for flexible application in different media and systems. We give a detailed description of the novel experimental setup and its measuring characteristics along with examples of application in different media and organisms to highlight its broad applicability, but also to discuss current limitations and challenges. The combined FIA-MDP approach for H2O2 quantification was used in different biological systems ranging from marine biology, using the model organism Exaiptasia pallida (light stress induced H2O2 release up to ~ 2.7 µM), over biomedical applications quantifying enzyme dynamics (glucose oxidase in a glucose solution producing up to ~ 60 µM H2O2 and the subsequent addition of catalase to monitor the H2O2 degradation process) and the ability of bacteria to modify their direct environment by regulating H2O2 concentrations in their surrounding media. This was shown by the bacteria Pseudomonas aeruginosa degrading ~ 18 µM background H2O2 in LB-broth. We also discuss advantages and current limitations of the FIA-MDP system, including a discussion of potential cross-sensitivity and interfering chemical species.

Pseudomonas aeruginosa biofilm hampers murine central wound healing by suppression of vascular epithelial growth factor.

Biofilm-infected wounds are clinically challenging. Vascular endothelial growth factor and host defence S100A8/A9 are crucial for wound healing but may be suppressed by biofilms. The natural course of Pseudomonas aeruginosa biofilm infection was compared in central and peripheral zones of burn-wounded, infection-susceptible BALB/c mice, which display delayed wound closure compared to C3H/HeN mice. Wounds were evaluated histopathologically 4, 7 or 10 days post-infection. Photoplanimetry evaluated necrotic areas. P. aeruginosa biofilm suppressed vascular endothelial growth factor levels centrally in BALB/c wounds but increased peripheral levels 4-7 days post-infection. Central zones of the burn wound displayed lower levels of central vascular endothelial growth factor as observed 4 and 7 days post-infection in BALB/c mice compared to their C3H/HeN counterparts. Biofilm suppressed early, centrally located S100A8/A9 in BALB/c and centrally and peripherally later on in C3H/HeN wounds as compared to uninfected mice. Peripheral polymorphonuclear-dominated inflammation and larger necrosis were observed in BALB/c wounds. In conclusion, P. aeruginosa biofilm modulates wounds by suppressing central, but inducing peripheral, vascular endothelial growth factor levels and reducing host response in wounds of BALB/c mice. This suppression is detrimental to the resolution of biofilm-infected necrosis.

The Inoculation Method Could Impact the Outcome of Microbiological Experiments.

For the past 150 years, bacteria have been investigated primarily in liquid batch cultures. Contrary to most expectations, these cultures are not homogeneous mixtures of single-cell bacteria, because free-floating bacterial aggregates eventually develop in most liquid batch cultures. These aggregates share characteristics with biofilms, such as increased antibiotic tolerance. We investigated how aggregates develop and what influences this development in liquid batch cultures of Pseudomonas aeruginosa We focused on how the method of inoculation affected aggregation by assessing aggregate frequency and size using confocal laser scanning microscopy. Several traditional methods of initiating an overnight bacterial culture, i.e., inoculation directly from frozen cultures, inoculation using agar-grown cells, or inoculation using cells grown in liquid cultures, were investigated. We discovered a direct link between the inoculation method and the size and frequency of biofilm aggregates in liquid batch cultures, with inoculation directly from a plate resulting in the most numerous and largest aggregates. These large aggregates had an overall impact on the cultures' subsequent tolerance toward tobramycin, indicating that the inoculation method has a profound impact on antibiotic tolerance. We also observed a mechanism whereby preformed aggregates recruited single cells from the surrounding culture in a "snowball effect," building up aggregated biomass in the culture. This recruitment was found to rely heavily on the exopolysaccharide Psl. Additionally, we found that both Escherichia coli and Staphylococcus aureus produced aggregates in liquid batch cultures. Our results stress the importance of inoculation consistency throughout experiments and the substantial impact aggregate development in liquid batch cultures may have on the outcomes of microbiological experiments.IMPORTANCE Pure liquid cultures are fundamental to the field of microbiological research. These cultures are normally thought of as homogeneous mixtures of single-cell bacteria; the present study shows that this is not always true. Bacteria may aggregate in these liquid cultures. The aggregation can be induced by the method chosen for inoculation. The presence of aggregates can significantly change the outcomes of experiments by altering the phenotype of the cultures. The study found a mechanism whereby preformed aggregates are able to recruit surrounding single cells in a form of snowball effect, creating more and larger aggregates in the cultures. Once formed, these aggregates are hard to remove. Aggregates in liquid cultures may be an immense unseen challenge for microbiologists.

The Consequences of Being in an Infectious Biofilm: Microenvironmental Conditions Governing Antibiotic Tolerance.

The main driver behind biofilm research is the desire to understand the mechanisms governing the antibiotic tolerance of biofilm-growing bacteria found in chronic bacterial infections. Rather than genetic traits, several physical and chemical traits of the biofilm have been shown to be attributable to antibiotic tolerance. During infection, bacteria in biofilms exhibit slow growth and a low metabolic state due to O2 limitation imposed by intense O2 consumption of polymorphonuclear leukocytes or metabolically active bacteria in the biofilm periphery. Due to variable O2 availability throughout the infection, pathogen growth can involve aerobic, microaerobic and anaerobic metabolism. This has serious implications for the antibiotic treatment of infections (e.g., in chronic wounds or in the chronic lung infection of cystic fibrosis patients), as antibiotics are usually optimized for aerobic, fast-growing bacteria. This review summarizes knowledge about the links between the microenvironment of biofilms in chronic infections and their tolerance against antibiotics.

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.

Microenvironmental characteristics and physiology of biofilms in chronic infections of CF patients are strongly affected by the host immune response.

In vitro studies of Pseudomonas aeruginosa and other pathogenic bacteria in biofilm aggregates have yielded detailed insight into their potential growth modes and metabolic flexibility under exposure to gradients of substrate and electron acceptor. However, the growth pattern of P. aeruginosa in chronic lung infections of cystic fibrosis (CF) patients is very different from what is observed in vitro, for example, in biofilms grown in flow chambers. Dense in vitro biofilms of P. aeruginosa exhibit rapid O2 depletion within <50-100 µm due to their own aerobic metabolism. In contrast, in vivo investigations show that P. aeruginosa persists in the chronically infected CF lung as relatively small cell aggregates that are surrounded by numerous PMNs, where the activity of PMNs is the major cause of O2 depletion rendering the P. aeruginosa aggregates anoxic. High levels of nitrate and nitrite enable P. aeruginosa to persist fueled by denitrification in the PMN-surrounded biofilm aggregates. This configuration creates a potentially long-term stable ecological niche for P. aeruginosa in the CF lung, which is largely governed by slow growth and anaerobic metabolism and enables persistence and resilience of this pathogen even under the recurring aggressive antimicrobial treatments of CF patients. As similar slow growth of other CF pathogens has recently been observed in endobronchial secretions, there is now a clear need for better in vitro models that simulate such in vivo growth patterns and anoxic microenvironments in order to help unravel the efficiency of existing or new antimicrobials targeting anaerobic metabolism in P. aeruginosa and other CF pathogens. We also advocate that host immune responses such as PMN-driven O2 depletion play a central role in the formation of anoxic microniches governing bacterial persistence in other chronic infections such as chronic wounds.

Development of a rechargeable optical hydrogen peroxide sensor - sensor design and biological application.

Hydrogen peroxide (H2O2) is an important member of the reactive oxygen species (ROS) family. Among ROS, H2O2 is considered the most long-lived and can accumulate inside and outside of cells, where it is involved in both vital (signaling) and deadly (toxic) reactions depending on its concentration. Quantifying H2O2 within biological samples is challenging and often not possible. Here we present a quasi-reversible fiber-optic sensor capable of measuring H2O2 concentrations ranging from 1-100 µM within different biological samples. Based on a Prussian blue/white redox cycle and a simple sensor recharging and readout strategy, H2O2 can be measured with high spatial (∼500 µm) and temporal (∼30 s) resolution. The sensor has a broad applicability both in complex environmental and biomedical systems, as demonstrated by (i) H2O2 concentration profile measurements in natural photosynthetic biofilms under light stress reaching H2O2 concentrations as high as 15 µM, and (ii) the quantification of the transient increase of the extracellular concentration of H2O2 during stimulation of neutrophils.

Role of Multicellular Aggregates in Biofilm Formation.

UNLABELLED: In traditional models ofin vitrobiofilm development, individual bacterial cells seed a surface, multiply, and mature into multicellular, three-dimensional structures. Much research has been devoted to elucidating the mechanisms governing the initial attachment of single cells to surfaces. However, in natural environments and during infection, bacterial cells tend to clump as multicellular aggregates, and biofilms can also slough off aggregates as a part of the dispersal process. This makes it likely that biofilms are often seeded by aggregates and single cells, yet how these aggregates impact biofilm initiation and development is not known. Here we use a combination of experimental and computational approaches to determine the relative fitness of single cells and preformed aggregates during early development ofPseudomonas aeruginosabiofilms. We find that the relative fitness of aggregates depends markedly on the density of surrounding single cells, i.e., the level of competition for growth resources. When competition between aggregates and single cells is low, an aggregate has a growth disadvantage because the aggregate interior has poor access to growth resources. However, if competition is high, aggregates exhibit higher fitness, because extending vertically above the surface gives cells at the top of aggregates better access to growth resources. Other advantages of seeding by aggregates, such as earlier switching to a biofilm-like phenotype and enhanced resilience toward antibiotics and immune response, may add to this ecological benefit. Our findings suggest that current models of biofilm formation should be reconsidered to incorporate the role of aggregates in biofilm initiation. IMPORTANCE: During the past decades, there has been a consensus around the model of development of a biofilm, involving attachment of single planktonic bacterial cells to a surface and the subsequent development of a mature biofilm. This study presents results that call for a modification of this rigorous model. We show how free floating biofilm aggregates can have a profound local effect on biofilm development when attaching to a surface. Our findings show that an aggregate landing on a surface will eventually outcompete the biofilm population arising from single cells attached around the aggregate and dominate the local biofilm development. These results point to a regime where preformed biofilm aggregates may have a fitness advantage over planktonic cells when it comes to accessing nutrients. Our findings add to the increasingly prominent comprehension that biofilm lifestyle is the default for bacteria and that planktonic single cells may be only a transition state at the most.

Increased bactericidal activity of colistin on Pseudomonas aeruginosa biofilms in anaerobic conditions.

Tolerance towards antibiotics of Pseudomonas aeruginosa biofilms is recognized as a major cause of therapeutic failure of chronic lung infection in cystic fibrosis (CF) patients. This lung infection is characterized by antibiotic-tolerant biofilms in mucus with zones of O2 depletion mainly due to polymorphonuclear leukocytic activity. In contrast to the main types of bactericidal antibiotics, it has not been possible to establish an association between the bactericidal effects of colistin and the production of detectable levels of OH ˙ on several strains of planktonic P. aeruginosa. Therefore, we propose that production of OH ˙ may not contribute significantly to the bactericidal activity of colistin on P. aeruginosa biofilm. Thus, we investigated the effect of colistin treatment on biofilm of wild-type PAO1, a catalase-deficient mutant (ΔkatA) and a colistin-resistant CF isolate cultured in microtiter plates in normoxic- or anoxic atmosphere with 1 mM nitrate. The killing of bacteria during colistin treatment was measured by CFU counts, and the OH⋅ formation was measured by 3(')-(p-hydroxylphenyl fluorescein) fluorescein (HPF) fluorescence. Validation of the assay was done by hydrogen peroxide treatment. OH⋅ formation was undetectable in aerobic PAO1 biofilms during 3 h of colistin treatment. Interestingly, we demonstrate increased susceptibility of P. aeruginosa biofilms towards colistin during anaerobic conditions. In fact, the maximum enhancement of killing by anaerobic conditions exceeded 2 logs using 4 mg L(-1) of colistin compared to killing at aerobic conditions.

Exhaled breath analysis using electronic nose in cystic fibrosis and primary ciliary dyskinesia patients with chronic pulmonary infections.

The current diagnostic work-up and monitoring of pulmonary infections may be perceived as invasive, is time consuming and expensive. In this explorative study, we investigated whether or not a non-invasive exhaled breath analysis using an electronic nose would discriminate between cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) with or without various well characterized chronic pulmonary infections. We recruited 64 patients with CF and 21 with PCD based on known chronic infection status. 21 healthy volunteers served as controls. An electronic nose was employed to analyze exhaled breath samples. Principal component reduction and discriminant analysis were used to construct internally cross-validated receiver operator characteristic (ROC) curves. Breath profiles of CF and PCD patients differed significantly from healthy controls p = 0.001 and p = 0.005, respectively. Profiles of CF patients having a chronic P. aeruginosa infection differed significantly from to non-chronically infected CF patients p = 0.044. We confirmed the previously established discriminative power of exhaled breath analysis in separation between healthy subjects and patients with CF or PCD. Furthermore, this method significantly discriminates CF patients suffering from a chronic pulmonary P. aeruginosa (PA) infection from CF patients without a chronic pulmonary infection. Further studies are needed for verification and to investigate the role of electronic nose technology in the very early diagnostic workup of pulmonary infections before the establishment of a chronic infection.

Plasmodium falciparum avoids change in erythrocytic surface expression of phagocytosis markers during inhibition of nitric oxide synthase activity.

Nitric oxide (NO) accumulates in Plasmodium falciparum-infected erythrocytes. It may be produced by a parasite NO synthase (NOS) or by nitrate reduction. The parasite's benefit of NO accumulation is not understood. We investigated if inhibiting the P. falciparum NOS with specific and unspecific NOS inhibitors led to a decrease in intraerythrocytic NO accumulation and if this was associated with a change in surface expression of the phagocytosis markers CD47 and phosphatidyl serine. The specific inducible NOS inhibitors l-canavanine and GW274150 dose-dependently decreased intraerythrocytic NO while l-NMMA (an unspecific NOS inhibitor) and caveolin-1 scaffolding domain peptide (a specific endothelial NOS inhibitor) did not affect NO levels. Phosphatidyl serine externalization markedly increased upon P. falciparum infection. l-canavanine did not modify this whereas caveolin-1 scaffolding domain peptide increased the fraction of phosphatidyl serine exposing cells significantly. The infection did not change the level of expression of neither total CD47 nor its oxidized form. Unrelated to NOS inhibition, incubation with caveolin-1 scaffolding domain peptide lead to a decrease in oxidized CD47. In conclusion, the data imply that NOS inhibitors decrease NO accumulation in P. falciparum-infected erythrocytes but this does not correlate with the level of two major erythrocytic phagocytosis markers.

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

Chronic Pseudomonas aeruginosa lung infection is the most severe complication in patients with cystic fibrosis (CF). The infection is characterized 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 of P. 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 (NO(-) 3) 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 NO(-) 3 and found a significant increase of growth when supplementing PAO1 and clinical isolates with ≥150 µM NO(-) 3 and 100 µM NO(-) 3, 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 NO(-) 3. Activation of denitrification could be achieved by supplementation with as little as 62.5 µM of NO(-) 3 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 NO(-) 3 followed by a transient increase of NO(-) 2. 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.

Polymorphonuclear leukocytes restrict growth of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients.

Cystic fibrosis (CF) patients have increased susceptibility to chronic lung infections by Pseudomonas aeruginosa, but the ecophysiology within the CF lung during infections is poorly understood. The aim of this study was to elucidate the in vivo growth physiology of P. aeruginosa within lungs of chronically infected CF patients. A novel, quantitative peptide nucleic acid (PNA) fluorescence in situ hybridization (PNA-FISH)-based method was used to estimate the in vivo growth rates of P. aeruginosa directly in lung tissue samples from CF patients and the growth rates of P. aeruginosa in infected lungs in a mouse model. The growth rate of P. aeruginosa within CF lungs did not correlate with the dimensions of bacterial aggregates but showed an inverse correlation to the concentration of polymorphonuclear leukocytes (PMNs) surrounding the bacteria. A growth-limiting effect on P. aeruginosa by PMNs was also observed in vitro, where this limitation was alleviated in the presence of the alternative electron acceptor nitrate. The finding that P. aeruginosa growth patterns correlate with the number of surrounding PMNs points to a bacteriostatic effect by PMNs via their strong O2 consumption, which slows the growth of P. aeruginosa in infected CF lungs. In support of this, the growth of P. aeruginosa was significantly higher in the respiratory airways than in the conducting airways of mice. These results indicate a complex host-pathogen interaction in chronic P. aeruginosa infection of the CF lung whereby PMNs slow the growth of the bacteria and render them less susceptible to antibiotic treatment while enabling them to persist by anaerobic respiration.

Formation of hydroxyl radicals contributes to the bactericidal activity of ciprofloxacin against Pseudomonas aeruginosa biofilms.

Antibiotic-tolerant, biofilm-forming Pseudomonas aeruginosa has long been recognized as a major cause of chronic lung infections of cystic fibrosis patients. The mechanisms involved in the activity of antibiotics on biofilm are not completely clear. We have investigated whether the proposed induction of cytotoxic hydroxyl radicals (OH˙) during antibiotic treatment of planktonically grown cells may contribute to action of the commonly used antibiotic ciprofloxacin on P. aeruginosa biofilms. For this purpose, WT PAO1, a catalase deficient ΔkatA and a ciprofloxacin resistant mutant of PAO1 (gyrA), were grown as biofilms in microtiter plates and treated with ciprofloxacin. Formation of OH˙ and total amount of reactive oxygen species (ROS) was measured and viability was estimated. Formation of OH˙ and total ROS in PAO1 biofilms treated with ciprofloxacin was shown but higher levels were measured in ΔkatA biofilms, and no ROS production was seen in the gyrA biofilms. Treatment with ciprofloxacin decreased the viability of PAO1 and ΔkatA biofilms but not of gyrA biofilms. Addition of thiourea, a OH˙ scavenger, decreased the OH˙ levels and killing of PAO1 biofilm. Our study shows that OH˙ is produced by P. aeruginosa biofilms treated with ciprofloxacin, which may contribute to the killing of biofilm subpopulations.

Pseudomonas aeruginosa biofilm aggravates skin inflammatory response in BALB/c mice in a novel chronic wound model.

Chronic wounds are presumed to persist in the inflammatory state, preventing healing. Emerging evidence indicates a clinical impact of bacterial biofilms in soft tissues, including Pseudomonas aeruginosa (PA) biofilms. To further investigate this, we developed a chronic PA biofilm wound infection model in C3H/HeN and BALB/c mice. The chronic wound was established by an injection of seaweed alginate-embedded P. aeruginosa PAO1 beneath a third-degree thermal lesion providing full thickness skin necrosis, as in human chronic wounds. Cultures revealed growth of PA, and both alginate with or without PAO1 generated a polymorphonuclear-dominated inflammation early after infection. However, both at days 4 and 7, there were a more acute polymorphonuclear-dominated and higher degree of inflammation in the PAO1 containing group (p < 0.05). Furthermore, PNA-FISH and supplemented DAPI staining showed bacteria organized in clusters, resembling biofilms, and inflammation located adjacent to the PA. The chronic wound infection showed a higher number of PAO1 in the BALB/c mice at day 4 after infection as compared to C3H/HeN mice (p < 0.006). In addition, a higher concentration of interleukin-1beta in the chronic wounds of BALB/c mice was observed at day 7 (p < 0.02), despite a similar number of bacteria in the two mouse strains. The present study succeeded in establishing a chronic PA biofilm infection in mice. The results showed an aggravating impact of local inflammation induced by PA biofilms. In conclusion, our findings indicate that improved infection control of chronic wounds reduces the inflammatory response and may improve healing.