Pitfalls Associated with Discriminating Mixed-Species Biofilms by Flow Cytometry.

Since biofilms are ubiquitous in different settings and act as sources of disease for humans, reliable methods to characterize and quantify these microbial communities are required. Numerous techniques have been employed, but most of them are unidirectional, labor intensive and time consuming. Although flow cytometry (FCM) can be a reliable choice to quickly provide a multiparametric analysis, there are still few applications on biofilms, and even less on the study of inter-kingdom communities. This work aimed to give insights into the application of FCM in order to more comprehensively analyze mixed-species biofilms, formed by different Pseudomonas aeruginosa and Candida albicans strains, before and after exposure to antimicrobials. For comparison purposes, biofilm culturability was also assessed determining colony-forming units. The results showed that some aspects, namely the microbial strain used, the morphological state of the cells and the biofilm matrix, make the accurate analysis of FCM data difficult. These aspects were even more challenging when double-species biofilms were being inspected, as they could engender data misinterpretations. The outcomes draw our attention towards the need to always take into consideration the characteristics of the biofilm samples to be analyzed through FCM, and undoubtedly link to the need for optimization of the processes tailored for each particular case study.

Unveiling the fate of adhering bacteria to antimicrobial surfaces: expression of resistance-associated genes and macrophage-mediated phagocytosis.

Since most antibacterial coatings reported to fight biomaterial-associated infections (BAI) fail in completely preventing bacterial colonization, it is crucial to know the impact of that small fraction of adhered bacteria in BAI recrudescence. This study aims to understand the fate of Staphylococcus aureus able to adhere to an antimicrobial coating previously developed, in terms of potential development of bacterial resistance and their macrophage-mediated phagocytosis. Antimicrobial coating comprised the co-immobilization of Palm peptide and DNase I onto polydimethylsiloxane. Expression of genes associated to resistance and virulence mechanisms showed that cells in contact with antimicrobial surfaces for a long period of 30 days, exhibit genes equally or less expressed, as compared to cells recovered from control surfaces. Recovered cells also exhibit the same susceptibility patterns, which strengthens the evidence of no resistance development. Remarkably, cells adhered to modified surfaces shows a reduced metabolic activity upon vancomycin treatment unlike the cells found on control surfaces, which can be identified as a clinical opportunity for prophylactically administration after implant surgery. Furthermore, results highlight that functionalization of PDMS with Palm and DNase I should not compromise the action of host immune cells. The overall results reinforce the potential of this antimicrobial strategy to fight BAI.

Polymicrobial Ventilator-Associated Pneumonia: Fighting In Vitro Candida albicans-Pseudomonas aeruginosa Biofilms with Antifungal-Antibacterial Combination Therapy.

The polymicrobial nature of ventilator-associated pneumonia (VAP) is now evident, with mixed bacterial-fungal biofilms colonizing the VAP endotracheal tube (ETT) surface. The microbial interplay within this infection may contribute for enhanced pathogenesis and exert impact towards antimicrobial therapy. Consequently, the high mortality/morbidity rates associated to VAP and the worldwide increase in antibiotic resistance has promoted the search for novel therapeutic strategies to fight VAP polymicrobial infections. Under this scope, this work aimed to assess the activity of mono- vs combinational antimicrobial therapy using one antibiotic (Polymyxin B; PolyB) and one antifungal (Amphotericin B; AmB) agent against polymicrobial biofilms of Pseudomonas aeruginosa and Candida albicans. The action of isolated antimicrobials was firstly evaluated in single- and polymicrobial cultures, with AmB being more effective against C. albicans and PolyB against P. aeruginosa. Mixed planktonic cultures required equal or higher antimicrobial concentrations. In biofilms, only PolyB at relatively high concentrations could reduce P. aeruginosa in both monospecies and polymicrobial populations, with C. albicans displaying only punctual disturbances. PolyB and AmB exhibited a synergistic effect against P. aeruginosa and C. albicans mixed planktonic cultures, but only high doses (256 mg L-1) of PolyB were able to eradicate polymicrobial biofilms, with P. aeruginosa showing loss of cultivability (but not viability) at 2 h post-treatment, whilst C. albicans only started to be inhibited after 14 h. In conclusion, combination therapy involving an antibiotic and an antifungal agent holds an attractive therapeutic option to treat severe bacterial-fungal polymicrobial infections. Nevertheless, optimization of antimicrobial doses and further clinical pharmacokinetics/pharmacodynamics and toxicodynamics studies underpinning the optimal use of these drugs are urgently required to improve therapy effectiveness and avoid reinfection.

Discriminating typical and atypical cystic fibrosis-related bacteria by multiplex PNA-FISH.

This study aims to report the development of peptide nucleic acid (PNA) probes to specifically detect the cystic fibrosis (CF)-associated traditional and atypical species Pseudomonas aeruginosa and Inquilinus limosus, respectively. PNA probes were designed in silico, developed and tested in smears prepared in phosphate-buffer saline (PBS), and in artificial sputum medium (ASM). A multiplex fluorescent in situ hybridization (FISH) approach using the designed probes was further validated in artificially contaminated clinical sputum samples and also applied in polymicrobial 24 h-old biofilms involving P. aeruginosa, I. limosus, and other CF-related bacteria. Both probes showed high predictive and experimental specificities and sensitivities. The multiplex PNA-FISH assay, associated with non-specific staining, was successfully adapted in the clinical samples and in biofilms of CF-related bacteria, allowing differentiating the community members and inferring about microbial-microbial interactions within the consortia. This study revealed the great potential of PNA-FISH as a diagnostic tool to discriminate between classical and less common CF-associated bacteria, being suitable to further describe species-dependent prevention strategies and deliver more effective target control therapeutics. Biotechnol. Bioeng. 2017;114: 355-367. © 2016 Wiley Periodicals, Inc.

Insights into Cystic Fibrosis Polymicrobial Consortia: The Role of Species Interactions in Biofilm Development, Phenotype, and Response to In-Use Antibiotics.

Cystic Fibrosis (CF) airways disease involves complex polymicrobial infections where different bacterial species can interact and influence each other and/or even interfere with the whole community. To gain insights into the role that interactions between Pseudomonas aeruginosa in co-culture with Staphylococcus aureus, Inquilinus limosus, and Stenotrophomonas maltophilia may play in infection, the reciprocal effect during biofilm formation and the response of dual biofilms toward ciprofloxacin under in vitro atmospheres with different oxygen availabilities were evaluated. Biofilm formation kinetics showed that the growth of S. aureus, I. limosus, and S. maltophilia was disturbed in the presence of P. aeruginosa, under both aerobic and anaerobic environments. On the other hand, under aerobic conditions, I. limosus led to a decrease in biofilm mass production by P. aeruginosa, although biofilm-cells viability remains unaltered. The interaction between S. maltophilia and P. aeruginosa positively influenced dual biofilm development by increasing its biomass. Compared with monocultures, biomass of P. aeruginosa+ S. aureus biofilms was significantly reduced by reciprocal interference. When grown in dual biofilms with P. aeruginosa, ciprofloxacin was less effective against S. aureus, I. limosus, and S. maltophilia, with increasing antibiotic doses leading to drastic inhibitions of P. aeruginosa cultivability. Therefore, P. aeruginosa might be responsible for the protection of the whole dual consortia against ciprofloxacin activity. Based on the overall data, it can be speculated that reciprocal interferences occur between the different bacterial species in CF lung, regardless the level of oxygen. The findings also suggest that alterations of bacterial behavior due to species interplay may be important for disease progression in CF infection.

The cystic fibrosis microbiome in an ecological perspective and its impact in antibiotic therapy.

The recent focus on the cystic fibrosis (CF) complex microbiome has led to the recognition that the microbes can interact between them and with the host immune system, affecting the disease progression and treatment routes. Although the main focus remains on the interactions between traditional pathogens, growing evidence supports the contribution and the role of emergent species. Understanding the mechanisms and the biological effects involved in polymicrobial interactions may be the key to improve effective therapies and also to define new strategies for disease control. This review focuses on the interactions between microbe-microbe and host-microbe, from an ecological point of view, discussing their impact on CF disease progression. There are increasing indications that these interactions impact the success of antimicrobial therapy. Consequently, a new approach where therapy is personalized to patients by taking into account their individual CF microbiome is suggested.

Microbiome in cystic fibrosis: Shaping polymicrobial interactions for advances in antibiotic therapy.

Recent molecular methodologies have demonstrated a complex microbial ecosystem in cystic fibrosis (CF) airways, with a wide array of uncommon microorganisms co-existing with the traditional pathogens. Although there are lines of evidence supporting the contribution of some of those emergent species for lung disease chronicity, clinical significance remains uncertain for most cases. A possible contribution for disease is likely to be related with the dynamic interactions established between microorganisms within the microbial community and with the host. If this is the case, management of CF will only be successful upon suitable and exhaustive modulation of such mixed ecological processes, which will also be useful to predict the effects of new therapeutic interventions.

Emergent bacteria in cystic fibrosis: in vitro biofilm formation and resilience under variable oxygen conditions.

Concurrent to conventional bacterial pathogens, unusual microbes are emerging from cystic fibrosis (CF) airways. Nonetheless, little is known about the contribution of these newly microbes to the resilience of CF-associated biofilms, particularly under variable-oxygen concentrations that are known to occur in vivo in the mucus of CF patients. Two CF-emergent bacterial species, Inquilinus limosus and Dolosigranulum pigrum, and the major pathogen Pseudomonas aeruginosa were studied in terms of biofilm development and antibiotic susceptibilities under in vitro atmospheres with different oxygen availabilities. All species were able to develop in vitro biofilms under different oxygen-available environments, with D. pigrum accumulating high amounts of biomass and respiratory activities. When established, biofilms were of difficult eradication, with antibiotics losing their effectiveness in comparison with the corresponding planktonic populations. Surprisingly, biofilms of each emergent organism displayed multidrug resistance under aerobic environments, enduring even in low-oxygen atmospheres. This study suggests a potential prospect on the impact of nonconventional organisms I. limosus and D. pigrum on CF lung infections, demonstrating capacity to adapt to biofilm mode of life under restricted-oxygen atmospheres resembling CF airways, which may ultimately endanger the efficacy of currently used antibiotic regimens.

Antimicrobial Pressure of Ciprofloxacin and Gentamicin on Biofilm Development by an Endoscope-Isolated Pseudomonas aeruginosa.

This work aims at characterizing endoscope biofilm-isolated (PAI) and reference strain P. aeruginosa (PA) adhesion, biofilm formation and sensitivity to antibiotics. The recovery ability of the biofilm-growing bacteria subjected to intermittent antibiotic pressure (ciprofloxacin (CIP) and gentamicin (GM)), as well as the development of resistance towards antibiotics and benzalkonium chloride (BC), were also determined. The capacity of both strains to develop biofilms was greatly impaired in the presence of CIP and GM. Sanitization was not complete allowing biofilm recovery after the intermittent cycles of antibiotic pressure. The environmental pressure exerted by CIP and GM did not develop P. aeruginosa resistance to antibiotics nor cross-resistance towards BC. However, data highlighted that none of the antimicrobials led to complete biofilm eradication, allowing the recovery of the remaining adhered population possibly due to the selection of persister cells. This feature may lead to biofilm recalcitrance, reinforcement of bacterial attachment, and recolonization of other sites.

Time to "go large" on biofilm research: advantages of an omics approach.

In nature, the biofilm mode of life is of great importance in the cell cycle for many microorganisms. Perhaps because of biofilm complexity and variability, the characterization of a given microbial system, in terms of biofilm formation potential, structure and associated physiological activity, in a large-scale, standardized and systematic manner has been hindered by the absence of high-throughput methods. This outlook is now starting to change as new methods involving the utilization of microtiter-plates and automated spectrophotometry and microscopy systems are being developed to perform large-scale testing of microbial biofilms. Here, we evaluate if the time is ripe to start an integrated omics approach, i.e., the generation and interrogation of large datasets, to biofilms--"biofomics". This omics approach would bring much needed insight into how biofilm formation ability is affected by a number of environmental, physiological and mutational factors and how these factors interplay between themselves in a standardized manner. This could then lead to the creation of a database where biofilm signatures are identified and interrogated. Nevertheless, and before embarking on such an enterprise, the selection of a versatile, robust, high-throughput biofilm growing device and of appropriate methods for biofilm analysis will have to be performed. Whether such device and analytical methods are already available, particularly for complex heterotrophic biofilms is, however, very debatable.

Antagonism between Bacillus cereus and Pseudomonas fluorescens in planktonic systems and in biofilms.

In the environment, many microorganisms coexist in communities competing for resources, and they are often associated as biofilms. The investigation of bacterial ecology and interactions may help to improve understanding of the ability of biofilms to persist. In this study, the behaviour of Bacillus cereus and Pseudomonas fluorescens in the planktonic and sessile states was compared. Planktonic tests were performed with single and dual species cultures in growth medium with and without supplemental FeCl3. B. cereus and P. fluorescens single cultures had equivalent growth behaviours. Also, when in co-culture under Fe-supplemented conditions, the bacteria coexisted and showed similar growth profiles. Under Fe limitation, 8 h after co-culture and over time, the number of viable B. cereus cells decreased compared with P. fluorescens. Spores were detected during the course of the experiment, but were not correlated with the decrease in the number of viable cells. This growth inhibitory effect was correlated with the release of metabolite molecules by P. fluorescens through Fe-dependent mechanisms. Biofilm studies were carried out with single and dual species using a continuous flow bioreactor rotating system with stainless steel (SS) substrata. Steady-state biofilms were exposed to a series of increasing shear stress forces. Analysis of the removal of dual species biofilms revealed that the outer layer was colonised mainly by B. cereus. This bacterium was able to grow in the outermost layers of the biofilm due to the inhibitory effect of P. fluorescens being decreased by the exposure of the cells to fresh culture medium. B. cereus also constituted the surface primary coloniser due to its favourable adhesion to SS. P. fluorescens was the main coloniser of the middle layers of the biofilm. Single and dual species biofilm removal data also revealed that B. cereus biofilms had the highest physical stability, followed by P. fluorescens biofilms. This study highlights the inadequacy of planktonic systems to mimic the behaviour of bacteria in biofilms and shows how the culturing system affects the action of antagonist metabolite molecules because dilution and consequent loss of activity occurred in continuously operating systems. Furthermore, the data demonstrate the biocontrol potential of P. fluorescens on the planktonic growth of B. cereus and the ability of the two species to coexist in a stratified biofilm structure.

The effects of a biocide and a surfactant on the detachment of Pseudomonas fluorescens from glass surfaces.

Application of antimicrobial chemicals is a general procedure in the cleaning and disinfection of food-contacting surfaces. Adhesion to glass surfaces and chemically induced detachment of Pseudomonas fluorescens ATCC 13525(T) were studied in situ, under flow conditions, in a well-controlled parallel plate flow chamber (PPFC). Ortho-phthalaldehyde (OPA) and cetyltrimethyl ammonium bromide (CTAB) were applied separately, at several concentrations, to attached bacteria and their subsequent detachment was monitored. Following treatments the remaining adhered bacteria were characterized in terms of viability and cell size. Simultaneously, the planktonic cell surface was characterized in order to correlate PPFC results with thermodynamic approaches for adhesion evaluation, and surface free energy of chemically treated cells with adhesion strength. About 2.8x10(6) cells/cm(2) adhered to the glass surface after 30 min of bacterial flow, although thermodynamic analyses evidenced unfavourable adhesion. The independent application of OPA and CTAB promoted bacterial detachment to a small extent (16% of total cells). The remaining adhering bacteria were totally non-viable for OPA> or =0.75 mM and CTAB> or =0.25 mM, showing a lack of correlation between bacterial viability and detachment. The cellular size decreased as attachment proceeded and with chemical treatment. Both chemicals altered the cell surface properties, increasing the cell-glass adhesion strength, and promoting the emergence of polar characteristics. The overall results emphasize that OPA and CTAB were markedly ineffective in removing glass-attached P. fluorescens, demonstrating that bacteria can be non-viable but remain strongly attached to the adhesion surface.

Sodium dodecyl sulfate allows the persistence and recovery of biofilms of Pseudomonas fluorescens formed under different hydrodynamic conditions.

The effect of the anionic surfactant sodium dodecyl sulfate (SDS) on Pseudomonas fluorescens biofilms was investigated using flow cell reactors with stainless steel substrata, under turbulent (Re = 5200) and laminar (Re = 2000) flow. Steady-state biofilms were exposed to SDS in single doses (0.5, 1, 3 and 7 mM) and biofilm respiratory activity and mass measured at 0, 3, 7 and 12 h after the SDS application. The effect of SDS on biofilm mechanical stability was assessed using a rotating bioreactor. Whilst high concentrations (7 mM) of SDS promoted significant biofilm inactivation, it did not significantly reduce biofouling. Turbulent and laminar flow-generated biofilms had comparable susceptibility to SDS application. Following SDS exposure, biofilms rapidly recovered over the following 12 h, achieving higher respiratory activity values than before treatment. This phenomenon of post-treatment recovery was more pronounced for turbulent flow-generated biofilms, with an increase in SDS concentration. The mechanical stability of the biofilms increased with surfactant application, except for SDS concentrations near the critical micellar concentration, as measured by biofilm removal due to an increase in external shear stress forces. The data suggest that although SDS exerts antimicrobial action against P. fluorescens biofilms, even if only partial and reversible, it had only limited antifouling efficacy, increasing biofilm mechanical stability at low concentrations and allowing significant and rapid recovery of turbulent flow-generated biofilms.

The effect of hydrodynamic conditions on the phenotype of Pseudomonas fluorescens biofilms.

This study investigated the phenotypic characteristics of monoculture P. fluorescens biofilms grown under turbulent and laminar flow, using flow cells reactors with stainless steel substrata. The cellular physiology and the overall biofilm activity, structure and composition were characterized, and compared, within hydrodynamically distinct conditions. The results indicate that turbulent flow-generated biofilm cells were significantly less extensive, with decreased metabolic activity and a lower protein and polysaccharides composition per cell than those from laminar flow-generated biofilms. The effect of flow regime did not cause significantly different outer membrane protein expression. From the analysis of biofilm activity, structure and composition, turbulent flow-generated biofilms were metabolically more active, had twice more mass per cm(2), and higher cellular density and protein content (mainly cellular) than laminar flow-generated biofilms. Conversely, laminar flow-generated biofilms presented higher total and matrix polysaccharide contents. Direct visualisation and scanning electron microscopy analysis showed that these different flows generate structurally different biofilms, corroborating the quantitative results. The combination of applied methods provided useful information regarding a broad spectrum of biofilm parameters, which can contribute to control and model biofilm processes.

Antimicrobial mechanisms of ortho-phthalaldehyde action.

Biocides generally have multiple biochemical targets. Such a feature easily entangles the analysis of the mechanisms of antimicrobial action. In this study, the action of the dialdehyde biocide ortho-phtalaldehyde (OPA), on bacteria, was investigated using the Gram-negative Pseudomonas fluorescens. The targets of the biocide action were studied using different bacterial physiological indices. The respiratory activity, membrane permeabilization, physico-chemical characterization of the bacterial surfaces, outer membrane proteins (OMP) expression, concomitant influence of pH, contact time and presence of bovine serum albumin (BSA) on respiratory activity, morphological changes and OPA-DNA interactions were assessed for different OPA concentrations. With the process conditions used, the minimum inhibitory concentration was 1500 mg/l, the concentration to promote total loss of bacterial culturability was 65 mg/l and the concentration needed to inactivate respiratory activity was 80 mg/l. These data are evidence that culturability and respiratory activity were markedly affected by the biocide. OPA lead, moreover, to a significant change in cell surface hydrophobicity and induced propidium iodide uptake. Such results suggest cytoplasmic membrane damage, although no release of ATP was detected. At pH 5, the bactericidal action of OPA was stronger, though not influenced by BSA presence. Nevertheless, at pH 9, BSA noticeably (p < 0.05) impaired biocide action. A time-dependent effect in OPA action was evident when contemplating respiratory activity variation, mainly for the lower exposure times. Scanning electron microscopy allowed to detect bacterial morphological changes, translated on cellular elongation, for OPA concentrations higher than 100 mg/l. Interferences at DNA level were, however, restricted to extreme biocide concentrations. The overall bactericidal events occurred without detectable OMP expression changes. In conclusion, the results indicated a sequence of events responsible for the antimicrobial action of OPA: it binds to membrane receptors due to cross-linkage; impairs the membrane functions allowing the biocide to enter through the permeabilized membrane; it interacts with intracellular reactive molecules, such as RNA, compromising the growth cycle of the cells and, at last, with DNA.