The non-attached biofilm aggregate.

Biofilms have conventionally been perceived as dense bacterial masses on surfaces, following the five-step model of development. Initial biofilm research focused on surface-attached formations, but detached aggregates have received increasing attention in the past decade due to their pivotal role in chronic infections. Understanding their nature sparked fervent discussions in biofilm conferences and scientific literature. This review consolidates current insights on non-attached aggregates, offering examples of their occurrence in nature and diseases. We discuss their formation and dispersion mechanisms, resilience to antibiotics and immune-responses, drawing parallels to surface-attached biofilms. Moreover, we outline available in vitro models for studying non-attached aggregates.

Pathological and microbiological impact of a gentamicin-loaded biocomposite following limited or extensive debridement in a porcine model of osteomyelitis.

AIMS: CERAMENT|G is an absorbable gentamicin-loaded biocomposite used as an on-site vehicle of antimicrobials for the treatment of chronic osteomyelitis. The purpose of the present study was to investigate the sole effect of CERAMENT|G, i.e. without additional systemic antimicrobial therapy, in relation to a limited or extensive debridement of osteomyelitis lesions in a porcine model. METHODS: Osteomyelitis was induced in nine pigs by inoculation of 104 colony-forming units (CFUs) of Staphylococcus aureus into a drill hole in the right tibia. After one week, the pigs were allocated into three groups. Group A (n = 3) received no treatment during the study period (19 days). Groups B (n = 3) and C (n = 3) received limited or extensive debridement seven days postinoculation, respectively, followed by injection of CERAMENT|G into the bone voids. The pigs were euthanized ten (Group C) and 12 (Group B) days after the intervention. RESULTS: All animals presented confirmatory signs of bone infection post-mortem. The estimated amount of inflammation was substantially greater in Groups A and B compared to Group C. In both Groups B and C, peptide nucleic acid fluorescence in situ hybridization (PNA FISH) of CERAMENT|G and surrounding bone tissue revealed bacteria embedded in an opaque matrix, i.e. within biofilm. In addition, in Group C, the maximal measured post-mortem gentamicin concentrations in CERAMENT|G and surrounding bone tissue samples were 16.6 µg/ml and 6.2 µg/ml, respectively. CONCLUSION: The present study demonstrates that CERAMENT|G cannot be used as a standalone alternative to extensive debridement or be used without the addition of systemic antimicrobials.Cite this article: Bone Joint Res 2020;9(7):394-401.

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.

In Vivo Gentamicin Susceptibility Test for Prevention of Bacterial Biofilms in Bone Tissue and on Implants.

The objective of this study was to set up an in vivo gentamicin susceptibility test for biofilm prevention in bone tissue and on implants. Twenty-five pigs were allocated to six groups. Pigs in group A (n = 6) were inoculated with saline. Pigs in groups B (n = 6), C (n = 3), D (n = 3), E (n = 3), and F (n = 4) were inoculated with 10 µl saline containing 104 CFU of Staphylococcus aureus Different concentrations based on the MIC of gentamicin for the specific strain were added to the 10-µl inoculum for groups C (160× MIC), D (1,600× MIC), E (16,000× MIC), and F (160,000× MIC). The inocula were injected into a predrilled tibial implant cavity, followed by insertion of a steel implant (2 by 15 mm). The pigs were euthanized after 5 days. In vitro, all the doses used were found to be bactericidal after up to 6 h. All implant cavities of pigs inoculated with bacteria and bacteria plus 160× MIC or 1,600× MIC of gentamicin were positive for S. aureus In animals in each of groups E (16,000× MIC) and F (160,000× MIC), 2/3 and 1/4 of the implant cavities were S. aureus positive, respectively. By grouping groups C and D (<10,000× MIC) and groups E and F (>10,000× MIC), a significant decrease in the number of implant-attached bacteria was seen only between the high-MIC-value group and group B. Histologically, it was demonstrated that 1,600×, 16,000×, and 160,000× MIC resulted in a peri-implant tissue reaction comparable to that in saline-inoculated animals. In vivo, the antimicrobial tolerance of the inoculated planktonic bacteria was increased by in vivo-specific factors of acute inflammation. This resulted in bacterial aggregation and biofilm formation, which further increased the gentamicin tolerance. Thus, susceptibility patterns in vitro might not reflect the actual in vivo susceptibility locally within a developing infectious area.

Bacterial Aggregates Establish at the Edges of Acute Epidermal Wounds.

Objective: The bacterial composition and distribution were evaluated in acute standardized epidermal wounds and uninjured skin by a molecular in situ technology benchmarked to conventional culturing. This was done to reveal whether bacterial biofilm is present in acute wounds. Approach: On the buttock of 26 healthy volunteers, 28 suction blisters were made and de-roofed. Four wounds were biopsied immediately after wounding, whereas the remaining 24 wounds were treated daily with sterile deionized water and covered with a moisture-retaining dressing. On day 4 post-wounding, swabs were obtained for culturing from the wounds and adjacent skin, and the wounds including adjacent skin were excised. Tissue sections were stained with peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) probes, counterstained by 4',6-diamidino-2-phenylindole, and evaluated by confocal laser scanning microscopy (CLSM). Results: No bacterial aggregates were detected at day 0. At day 4, coagulase-negative staphylococci (CoNS) were the sole bacteria identified by CLSM/PNA-FISH and culturing. CoNS was isolated from 78% of the wound swabs and 48% of the skin swabs. Bacterial aggregates (5-150 µm) were detected by PNA-FISH/CLSM in the split stratum corneum and fibrin deposits at the wound edges and in the stratum corneum and the hair follicles of the adjacent skin. The bacterial aggregates were more common (p = 0.0084) and larger (p = 0.0083) at wound edges than in the adjacent skin. Innovation: Bacterial aggregates can establish in all wound types and may have clinical significance in acute wounds. Conclusion: Bacterial aggregates were observed at the edges of acute epidermal wounds, indicating initiated establishment of a biofilm.

Bacterial biofilms: a possible mechanism for chronic infection in patients with lumbar disc herniation - a prospective proof-of-concept study using fluorescence in situ hybridization.

A relationship has been suggested between lumbar disc herniation (LDH) and chronic bacterial infection frequently involving Propionibacterium acnes, which is known to cause chronic infection through the formation of biofilm aggregates. The objective of the study was to assess whether a disc infection involving biofilm formation is present in patients with LDH. A total of 51 LDH patients and 14 controls were included. Bacterial DNA was detected by real-time polymerase chain reaction (PCR) in 16/51 samples in the LDH group and 7/14 controls (p = 0.215). Sequencing identified bacteria in 9/16 and 6/7 PCR positive samples in the LDH and control groups, respectively. All samples were stained using fluorescence in situ hybridization (FISH) and examined by confocal laser scanning microscopy. Microscopy demonstrated tissue-embedded bacterial aggregates with host inflammatory cells in 7/51 LDH patients and no controls. The presence of both bacterial aggregates and inflammatory cells suggests a chronic infection in a subset of LDH patients. The finding of bacterial 16S rDNA in both LDH and control disc tissue highlights the importance of microscopic observation to discriminate infection vs contamination. Our findings may have therapeutic implications, as the treatment of biofilm infections is different and more challenging than traditional infections.

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.

The Pseudomonas aeruginosa PSL Polysaccharide Is a Social but Noncheatable Trait in Biofilms.

Extracellular polysaccharides are compounds secreted by microorganisms into the surrounding environment, and they are important for surface attachment and maintaining structural integrity within biofilms. The social nature of many extracellular polysaccharides remains unclear, and it has been suggested that they could function as either cooperative public goods or as traits that provide a competitive advantage. Here, we empirically tested the cooperative nature of the PSL polysaccharide, which is crucial for the formation of biofilms in Pseudomonas aeruginosa We show that (i) PSL is not metabolically costly to produce; (ii) PSL provides population-level benefits in biofilms, for both growth and antibiotic tolerance; (iii) the benefits of PSL production are social and are shared with other cells; (iv) the benefits of PSL production appear to be preferentially directed toward cells which produce PSL; (v) cells which do not produce PSL are unable to successfully exploit cells which produce PSL. Taken together, this suggests that PSL is a social but relatively nonexploitable trait and that growth within biofilms selects for PSL-producing strains, even when multiple strains are on a patch (low relatedness at the patch level).IMPORTANCE Many studies have shown that bacterial traits, such as siderophores and quorum sensing, are social in nature. This has led to an impression that secreted traits act as public goods, which are costly to produce but benefit both the producing cell and its surrounding neighbors. Theories and subsequent experiments have shown that such traits are exploitable by asocial cheats, but we show here that this does not always hold true. We demonstrate that the Pseudomonas aeruginosa exopolysaccharide PSL provides social benefits to populations but that it is nonexploitable, because most of the fitness benefits accrue to PSL-producing cells. Our work builds on an increasing body of work showing that secreted traits can have both private and public benefits to cells.

Phage Inhibit Pathogen Dissemination by Targeting Bacterial Migrants in a Chronic Infection Model.

The microbial communities inhabiting chronic infections are often composed of spatially organized micrometer-sized, highly dense aggregates. It has recently been hypothesized that aggregates are responsible for the high tolerance of chronic infections to host immune functions and antimicrobial therapies. Little is currently known regarding the mechanisms controlling aggregate formation and antimicrobial tolerance primarily because of the lack of robust, biologically relevant experimental systems that promote natural aggregate formation. Here, we developed an in vitro model based on chronic Pseudomonas aeruginosa infection of the cystic fibrosis (CF) lung. This model utilizes a synthetic sputum medium that readily promotes the formation of P. aeruginosa aggregates with sizes similar to those observed in human CF lung tissue. Using high-resolution imaging, we exploited this model to elucidate the life history of P. aeruginosa and the mechanisms that this bacterium utilizes to tolerate antimicrobials, specifically, bacteriophage. In the early stages of growth in synthetic sputum, planktonic cells form aggregates that increase in size over time by expansion. In later growth, migrant cells disperse from aggregates and colonize new areas, seeding new aggregates. When added simultaneously with phage, P. aeruginosa was readily killed and aggregates were unable to form. When added after initial aggregate formation, phage were unable to eliminate all of the aggregates because of exopolysaccharide production; however, seeding of new aggregates by dispersed migrants was inhibited. We propose a model in which aggregates provide a mechanism that allows P. aeruginosa to tolerate phage therapy during chronic infection without the need for genetic mutation.IMPORTANCE Bacteria in chronic infections often reside in communities composed of micrometer-sized, highly dense aggregates. A primary challenge for studying aggregates has been the lack of laboratory systems that promote natural aggregate formation in relevant environments. Here, we developed a growth medium that mimics chronic lung infection and promotes natural aggregate formation by the bacterium Pseudomonas aeruginosa High-resolution, single-cell microscopy allowed us to characterize P. aeruginosa's life history-seeding, aggregate formation, and dispersal-in this medium. Our results reveal that this bacterium readily forms aggregates that release migrants to colonize new areas. We also show that aggregates allow P. aeruginosa to tolerate therapeutic bacteriophage addition, although this treatment limits P. aeruginosa dissemination by targeting migrants.

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.

Phenotypic shift in Pseudomonas aeruginosa populations from cystic fibrosis lungs after 2-week antipseudomonal treatment.

BACKGROUND: The influence of suppressive therapy on the different P. aeruginosa phenotypes harbored in the lungs of cystic fibrosis (CF) patients remains unclear. Our aim was to investigate the phenotypic changes (mucoidy, hypermutability, antibiotic resistance, transcriptomic profiles and biofilm) in P. aeruginosa populations before and after a 2-week course of suppressive antimicrobial therapy in chronically infected CF patients in Denmark. MATERIAL AND METHODS: Prospective observational clinical study. Sputum samples were assessed before and after treatment for P. aeruginosa, with regard to: a) colony-forming units (CFU/mL), b) frequency of mucoids and non-mucoids, c) resistance pattern to anti-pseudomonal drugs, d) hypermutability, e) transcriptomic profiles, and f) presence of biofilms. RESULTS: We collected 23 sputum samples (12 before antibiotic treatment and 11 after) and 77 P. aeruginosa from different CF patients. After treatment, the P. aeruginosa burden diminished but antimicrobial resistance to aztreonam, tobramycin and ceftazidime rose; non-mucoid phenotypes presented increased resistance to colistin, tobramycin, meropenem, and ciprofloxacin, and hypermutable phenotypes to ciprofloxacin. In spite of biofilm persistence, a down-regulation of genes involved in denitrification was detected. CONCLUSION: A 2-week course of suppressive therapy reduces P. aeruginosa lung colonization and influences nitrogen metabolism genes, but also promotes antimicrobial resistance while P. aeruginosa persists in biofilms.

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.

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.

Autofluorescence in samples obtained from chronic biofilm infections--"all that glitters is not gold".

When looking at tissue sections of ex vivo samples, autofluorescence can be a major cause of artifacts and misinterpretations. We here reiterate evidence that autofluorescing granules, often hemosiderin but also ceroid or mucinogen granules, are severe obstacles when imaging and diagnosing biofilm infections through fluorescent imaging techniques. We used confocal laser scanning microscopy with spectral analysis for autofluorescence detection as well as standard histological stains in order to identify the culprit and show that these granules might very well be mistaken for bacterial biofilms. Furthermore, we hypothesize that the increased amount of autofluorescing granules may be a consequence of prolonged inflammation as a consequence of chronic biofilm infections.

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.

The microorganisms in chronically infected end-stage and non-end-stage cystic fibrosis patients.

Patients suffering from cystic fibrosis (CF) develop chronic lung infections because of highly viscous mucus, where bacteria can form biofilms. In this study, we investigated the microorganisms present in the lungs of end-stage and non-end-stage patients using standard culturing techniques and molecular methods. Tissue and sputum samples (n = 34) from explanted lungs of five end-stage patients were examined along with routine expectorates (n = 15) from 13 patients with non-end-stage CF, representing earlier stages of chronic lung infections. Previously, using peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH), we have shown that Pseudomonas aeruginosa was the sole pathogen in end-stage CF lungs (Pediatr Pulmonol 2009, 44: 547). In this study, this tendency was supported by the results of real-time PCR, confirming previous results obtained by standard culturing and 16S rRNA gene analysis (J Clin Microbiol 2011, 49: 4352). Conversely, the non-end-stage patients were found to harbor several species by culturing. PNA FISH confirmed heterogeneous microbiota and showed that the bacteria were located in monospecies aggregates with no apparent physical interaction between the different microcolonies. In conclusion, standard culturing identifies the dominating pathogens, which seem to reside in monospecies microcolonies. The possibility of signaling between the distinct microcolonies still has to be verified and elucidated.