Kasper Nørskov Kragh

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.

Role of Multicellular Aggregates in Biofilm Formation

ABSTRACT In traditional models of in vitro biofilm 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 of Pseudomonas aeruginosa biofilms. 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. 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.

Polymorphonuclear Leukocytes Restrict Growth of Pseudomonas aeruginosa in the Lungs of Cystic Fibrosis Patients

ABSTRACT 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.

The Limitations of In Vitro Experimentation in Understanding Biofilms and Chronic Infection

We have become increasingly aware that, during infection, pathogenic bacteria often grow in multicellular biofilms that are often highly resistant to antibacterial strategies. In order to understand how biofilms form and contribute to infection, many research groups around the world have heavily used in vitro biofilm systems such as microtitre plate assays and flow cells. Whilst these methods have greatly increased our understanding of the biology of biofilms, it is becoming increasingly apparent that many of our in vitro methods do not accurately represent in vivo conditions. Here we present a systematic review of the most widely used in vitro biofilm systems, and we discuss why they are not always representative of the in vivo biofilms found in chronic infections. We present examples of methods that will help us to bridge the gap between in vitro and in vivo biofilm work so that we can ultimately use our benchside data to improve bedside treatment.

True Microbiota Involved in Chronic Lung Infection of Cystic Fibrosis Patients Found by Culturing and 16S rRNA Gene Analysis

ABSTRACT Patients suffering from cystic fibrosis (CF) develop chronic lung infection. In this study, we investigated the microorganisms present in transplanted CF lungs (n = 5) by standard culturing and 16S rRNA gene analysis. A correspondence between culturing and the molecular methods was observed. In conclusion, standard culturing seems reliable for the identification of the dominating pathogens.

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.

Bacterial biofilm formation and treatment in soft tissue fillers.

Injection of soft tissue fillers plays an important role in facial reconstruction and esthetic treatments such as cosmetic surgery for lip augmentation and wrinkle smoothening. Adverse events are an increasing problem, and recently, it has been suggested that bacteria are the cause of a vast fraction these. We developed a novel mouse model and evaluated hyaluronic acid gel, calcium hydroxyl apatite microspheres, and polyacrylamide hydrogel for their potential for sustaining bacterial infections and their possible treatments. We were able to culture Pseudomonas aeruginosa, Staphylococcus epidermidis, and Probionibacterium acnes in all three gels. When contaminated gels were left for 7 days in a mouse model, we found sustainment of bacterial infection with the permanent gel, less with the semi-permanent gel, and no growth within the temporary gel. Evaluation of treatment strategies showed that once the bacteria had settled (into biofilms) within the gels, even successive treatments with high concentrations of relevant antibiotics were not effective. Our data substantiate bacteria as a cause of adverse reactions reported when using tissue fillers, and the sustainability of these infections appears to depend on longevity of the gel. Most importantly, the infections are resistant to antibiotics once established but can be prevented using prophylactic antibiotics.

Chronic pulmonary disease with Mycobacterium abscessus complex is a biofilm infection

Direct evidence of Mycobacterium abscessus complex (MABSC) biofilm in the human lung has not previously been demonstrated. Biofilms are microcolonies of bacteria, imbedded in extracellular matrix, providing stability and tolerance to antibiotics and the bodys innate and adaptive defences [1]. This mode of growth is an inherent feature of chronic infections and is particularly well studied for Pseudomonas aeruginosa and other Gram-negative infections [1], but also some Staphylococcus aureus infections [2]. Mycobacterial infections have also been shown to be capable of biofilm formation, most notably Mycobacterium tuberculosis (tuberculosis), which under the right conditions, can self-assemble into highly organised matrix-encapsulated biofilm [3]. Among the nontuberculous mycobacteria (NTM), Mycobacterium avium complex (MAC) and the rapidly growing mycobacteria, including MABSC, have been shown to grow as biofilms either in vitro or in environmental reservoirs [4, 5], but in vivo conditions have not been studied. MABSC is an emerging threat to patients with cystic fibrosis [6], who become infected at an early age and deteriorate clinically [7] as the persistent infection causes inflammation and tissue damage. We wanted to explore how MABSC grows in the antibiotic-rich, end-stage lungs of patients with cystic fibrosis. The aim was to describe the localisation and growth patterns of MABSC in vivo from freshly explanted lungs of patients with cystic fibrosis and a history of MASBC. We simultaneously performed histological and mycobacterial sampling from the same areas, from multiple pulmonary sites. Mycobacterial culture was performed by inoculation on at least one solid (Middlebrooke 7H10 or Löwenstein–Jensen slopes; SSI Diagnostica, Hilleroed, Denmark) and in one liquid culture medium (BACTEC 12B or MGIT; Becton Dickinson Microbiology Systems, Sparks, MD, USA), and a reverse hybridisation DNA assay was performed (InnoLiPA; Fujirebio Europe, Brondby, Denmark), as previously described [7]. Culture morphology was determined by direct visual inspection of colonies and control microscopy used Ziehl–Neelsen staining. Concomitant culture for Gram-negative and -positive bacteria was also performed. Patient files were reviewed to determine the clinical course of their end-stage lung disease and their history of other chronic bacterial infections. The NTM collection and clinical data collection was approved by the Committee on Health Research Ethics in the Capital Region of Denmark (Copenhagen, Denmark) (H-3-2012-098). The explanted lungs were collected by the transplantation team, microbiological samples were sent for NTM culture and histological samples were transferred to 4% formaldehyde before further preparation for microscopic investigation. The biopsy material was embedded in paraffin, cut into 4-μm sections and mounted on glass slides. Prior to microscopy, paraffin was removed and the tissue sections were analysed by means of conventional haematoxylin and eosin (H&E) staining, Ziehl–Neelsen staining (acid-fast stain), auramine dye (fluorescent stain), and fluorescent in situ hybridisation (FISH) with peptide nucleic acid (PNA) probes: a uniBac probe (red) and a NTM-specific probe (green) (AdvanDx, Inc., Woburn, MA, USA). We used 4′,6′-diamidino-2-phenylindole (Vector Laboratories) as a counterstain for DNA. Microscopic observations were performed with Zeiss 710 Confocal Laser Scanning Microscope (Leica Microsystems, Mannheim, Germany). Mycobacterium abscessus complex can establish a biofilm infection in the lung, which has implications for treatment http://ow.ly/RSjGZ

Shaping the Growth Behaviour of Biofilms Initiated from Bacterial Aggregates

Bacterial biofilms are usually assumed to originate from individual cells deposited on a surface. However, many biofilm-forming bacteria tend to aggregate in the planktonic phase so that it is possible that many natural and infectious biofilms originate wholly or partially from pre-formed cell aggregates. Here, we use agent-based computer simulations to investigate the role of pre-formed aggregates in biofilm development. Focusing on the initial shape the aggregate forms on the surface, we find that the degree of spreading of an aggregate on a surface can play an important role in determining its eventual fate during biofilm development. Specifically, initially spread aggregates perform better when competition with surrounding unaggregated bacterial cells is low, while initially rounded aggregates perform better when competition with surrounding unaggregated cells is high. These contrasting outcomes are governed by a trade-off between aggregate surface area and height. Our results provide new insight into biofilm formation and development, and reveal new factors that may be at play in the social evolution of biofilm communities.

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

ABSTRACT 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. IMPORTANCE Pseudomonas 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.