Biofilm Formation by Campylobacter jejuni Is Increased under Aerobic Conditions

The microaerophilic human pathogen Campylobacter jejuni is the leading cause of food-borne bacterial gastroenteritis in the developed world. During transmission through the food chain and the environment, the organism must survive stressful environmental conditions, particularly high oxygen levels. Biofilm formation has been suggested to play a role in the environmental survival of this organism. In this work we show that C. jejuni NCTC 11168 biofilms developed more rapidly under environmental and food-chain-relevant aerobic conditions (20% O₂) than under microaerobic conditions (5% O₂, 10% CO₂), although final levels of biofilms were comparable after 3 days. Staining of biofilms with Congo red gave results similar to those obtained with the commonly used crystal violet staining. The level of biofilm formation by nonmotile aflagellate strains was lower than that observed for the motile flagellated strain but nonetheless increased under aerobic conditions, suggesting the presence of flagellum-dependent and flagellum-independent mechanisms of biofilm formation in C. jejuni. Moreover, preformed biofilms shed high numbers of viable C. jejuni cells into the culture supernatant independently of the oxygen concentration, suggesting a continuous passive release of cells into the medium rather than a condition-specific active mechanism of dispersal. We conclude that under aerobic or stressful conditions, C. jejuni adapts to a biofilm lifestyle, allowing survival under detrimental conditions, and that such a biofilm can function as a reservoir of viable planktonic cells. The increased level of biofilm formation under aerobic conditions is likely to be an adaptation contributing to the zoonotic lifestyle of C. jejuni.Infection with Campylobacter jejuni is the leading cause of food-borne bacterial gastroenteritis in the developed world and is often associated with the consumption of undercooked poultry products (19). The United Kingdom Health Protection Agency reported more than 45,000 laboratory-confirmed cases for England and Wales in 2006 alone, although this is thought to be a 5- to 10-fold underestimation of the total number of community incidents (20, 43). The symptoms associated with C. jejuni infection usually last between 2 and 5 days and include diarrhea, vomiting, and stomach pains. Sequelae of C. jejuni infection include more-serious autoimmune diseases, such as Guillain-Barré syndrome, Miller-Fisher syndrome (18), and reactive arthritis (15).Poultry represents a major natural reservoir for C. jejuni, since the organism is usually considered to be a commensal and can reach densities as high as 1 × 10⁸ CFU g of cecal contents⁻¹ (35). As a result, large numbers of bacteria are shed via feces into the environment, and consequently, C. jejuni can spread rapidly through a flock of birds in a broiler house (1). While well adapted to life in the avian host, C. jejuni must survive during transit between hosts and on food products under stressful storage conditions, including high and low temperatures and atmospheric oxygen levels. The organism must therefore have mechanisms to protect itself from unfavorable conditions.Biofilm formation is a well-characterized bacterial mode of growth and survival, where the surface-attached and matrix-encased bacteria are protected from stressful environmental conditions, such as UV radiation, predation, and desiccation (7, 8, 28). Bacteria in biofilms are also known to be >1,000-fold more resistant to disinfectants and antimicrobials than their planktonic counterparts (11). Several reports have now shown that Campylobacter species are capable of forming a monospecies biofilm (21, 22) and can colonize a preexisting biofilm (14). Biofilm formation can be demonstrated under laboratory conditions, and environmental biofilms, from poultry-rearing facilities, have been shown to contain Campylobacter (5, 32, 44). Campylobacter biofilms allow the organism to survive up to twice as long under atmospheric conditions (2, 21) and in water systems (27).Molecular understanding of biofilm formation by Campylobacter is still in its infancy, although there is evidence for the role of flagella and gene regulation in biofilm formation. Indeed, a flaAB mutant shows reduced biofilm formation (34); mutants defective in flagellar modification (cj1337) and assembly (fliS) are defective in adhering to glass surfaces (21); and a proteomic study of biofilm-grown cells shows increased levels of motility-associated proteins, including FlaA, FlaB, FliD, FlgG, and FlgG2 (22). Flagella are also implicated in adhesion and in biofilm formation and development in other bacterial species, including Aeromonas, Vibrio, Yersinia, and Pseudomonas species (3, 23, 24, 31, 42).Previous studies of Campylobacter biofilms have focused mostly on biofilm formation under standard microaerobic laboratory conditions. In this work we have examined the formation of biofilms by motile and nonmotile C. jejuni strains under atmospheric conditions that are relevant to the survival of this organism in a commercial context of environmental and food-based transmission.     

Comparison of Switching and Biofilm Formation between MTL-Homozygous Strains of Candida albicans and Candida dubliniensis

Candida albicans and Candida dubliniensis are highly related species that share the same main developmental programs. In C. albicans, it has been demonstrated that the biofilms formed by strains heterozygous and homozygous at the mating type locus (MTL) differ functionally, but studies rarely identify the MTL configuration. This becomes a particular problem in studies of C. dubliniensis, given that one-third of natural strains are MTL homozygous. For that reason, we have analyzed MTL-homozygous strains of C. dubliniensis for their capacity to switch from white to opaque, the stability of the opaque phenotype, CO₂ induction of switching, pheromone induction of adhesion, the effects of minority opaque cells on biofilm thickness and dry weight, and biofilm architecture in comparison with C. albicans. Our results reveal that C. dubliniensis strains switch to opaque at lower average frequencies, exhibit a far lower level of opaque phase stability, are not stimulated to switch by high CO₂, exhibit more variability in biofilm architecture, and most notably, form mature biofilms composed predominately of pseudohyphae rather than true hyphae. Therefore, while several traits of MTL-homozygous strains of C. dubliniensis appear to be degenerating or have been lost, others, most notably several related to biofilm formation, have been conserved. Within this context, the possibility is considered that C. dubliniensis is transitioning from a hypha-dominated to a pseudohypha-dominated biofilm and that aspects of C. dubliniensis colonization may provide insights into the selective pressures that are involved.     

Prevention of Candida albicans Biofilm Formation by Covalently Bound Dimethylaminoethylmethacrylate and Polyethylenimine

Candida albicans biofilms are a major cause of voice prosthesis deterioration in laryngectomized patients. The aim of this study was to produce a surface capable of inhibiting C. albicans biofilm formation. Dimethylaminoethylmethacrylate (DMAEMA) and polyethylenimine (PEI) moieties were covalently bound to the surface of polydimethylsiloxane (PDMS) or polymethylmethacrylate (PMMA) and subsequently quaternized. Physicochemical characterization of the grafted surfaces was carried out and their effect on C. albicans cell numbers was assessed using a modified Robbins device to grow the biofilms. Covalently bound quaternized polyDMAEMA (polyDMAEMAq) and PEI (PEIq) inhibited biofilm growth, with reductions up to 92%. Our approach may show promise for future application in medical devices such as catheters and prostheses.     

Inhibition of Candida albicans Biofilm Formation by Farnesol, a Quorum-Sensing Molecule

Farnesol is a quorum-sensing molecule that inhibits filamentation in Candida albicans. Both filamentation and quorum sensing are deemed to be important factors in C. albicans biofilm development. Here we examined the effect of farnesol on C. albicans biofilm formation. C. albicans adherent cell populations (after 0, 1, 2, and 4 h of adherence) and preformed biofilms (24 h) were treated with various concentrations of farnesol (0, 3, 30, and 300 μM) and incubated at 37°C for 24 h. The extent and characteristics of biofilm formation were then assessed microscopically and with a semiquantitative colorimetric technique based on the use of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide. The results indicated that the effect of farnesol was dependent on the concentration of this compound and the initial adherence time, and preincubation with 300 μM farnesol completely inhibited biofilm formation. Supernatant media recovered from mature biofilms inhibited the ability of planktonic C. albicans to form filaments, indicating that a morphogenetic autoregulatory compound is produced in situ in biofilms. Northern blot analysis of RNA extracted from cells in biofilms indicated that the levels of expression of HWP1, encoding a hypha-specific wall protein, were decreased in farnesol-treated biofilms compared to the levels in controls. Our results indicate that farnesol acts as a naturally occurring quorum-sensing molecule which inhibits biofilm formation, and we discuss its potential for further development and use as a novel therapeutic agent.     

In Vitro Effect of Amphotericin B on Candida albicans,Candida glabrata and Candida parapsilosis Biofilm Formation

Candida spp. biofilm is considered highly resistant to conventional antifungals. The aim of this study was to investigate the in vitro effect of amphotericin B on Candida spp. biofilms at different stages of maturation. We investigated the activity of amphotericin B against 78 clinical isolates of Candida spp., representing three species, growing as planktonic and sessile cells, by a widely accepted broth microdilution method. The in vitro effect on sessile cell viability was evaluated by MTT reduction assay. All examined strains were susceptible to amphotericin B when grown as free-living cells. At the early stages of biofilm maturation 96.7–100.0 % strains, depending on species, displayed amphotericin B sessile minimal inhibitory concentration (SMIC) ≤1 μg/mL. Mature Candida spp. biofilm of 32.1–90.0 % strains displayed amphotericin B SMIC ≤1 μg/mL. Based on these results, amphotericin B displays species- and strain-depending activity against Candida spp. biofilms.     

Production of Nitric Oxide in Loam Under Aerobic and Anaerobic Conditions

Measurements of gas flow through soil columns of loam from Kjettslinge, Uppland, Sweden, gave average NO production rates of 0.06 ± 0.01 ng of NO N g of soil⁻¹ min⁻¹ in aerobic conditions and 3.7 ± 0.6 ng of NO N g of soil⁻¹ min⁻¹ in anaerobic conditions at 25°C. Approximately 30% of the NO₃⁻ loss in anaerobic conditions was as NO. In aerobic conditions an equilibrium concentration for NO was found. Above this concentration there was uptake of NO. Autoclaved samples indicated that less than 10% of the NO production was abiological, and there was no abiological NO uptake. The NO production reached anaerobic rates at soil O₂ levels between 0.5 and 0.05%.     

Streptococcus mutans Can Modulate Biofilm Formation and Attenuate the Virulence of Candida albicans

Streptococcus mutans and Candida albicans are found together in the oral biofilms on dental surfaces, but little is known about the ecological interactions between these species. Here, we studied the effects of S. mutans UA159 on the growth and pathogencity of C. albicans. Initially, the effects of S. mutans on the biofilm formation and morphogenesis of C. albicans were tested in vitro. Next, we investigate the influence of S. mutans on pathogenicity of C. albicans using in vivo host models, in which the experimental candidiasis was induced in G. mellonella larvae and analyzed by survival curves, C. albicans count in hemolymph, and quantification of hyphae in the host tissues. In all the tests, we evaluated the direct effects of S. mutans cells, as well as the indirect effects of the subproducts secreted by this microorganism using a bacterial culture filtrate. The in vitro analysis showed that S. mutans cells favored biofilm formation by C. albicans. However, a reduction in biofilm viable cells and inhibition of hyphal growth was observed when C. albicans was in contact with the S. mutans culture filtrate. In the in vivo study, injection of S. mutans cells or S. mutans culture filtrate into G. mellonella larvae infected with C. albicans increased the survival of these animals. Furthermore, a reduction in hyphal formation was observed in larval tissues when C. albicans was associated with S. mutans culture filtrate. These findings suggest that S. mutans can secrete subproducts capable to inhibit the biofilm formation, morphogenesis and pathogenicity of C. albicans, attenuating the experimental candidiasis in G. mellonella model.     

Swarming Characteristics of Proteus mirabilis Under Anaerobic and Aerobic Conditions

Nutrients have a pronounced effect on the growth and swarming behaviour of Proteus mirabilis 7002. Iron, zinc, amino acids, and dioxygen are important for rapid growth and normal swarming. Anaerobically grown cultures of P. mirabilis 7002 were unable to swarm on anaerobically maintained rich nutrient agar. Upon exposure to aerobic conditions, P. mirabilis 7002 resumed swarming behaviour. Scanning electron microscopy was used to demonstrate the presence of community organization and mature rafts during normal swarming. These results support the importance of dioxygen and redox status in cell differentiation.     

Anaerobic Growth of Purple Nonsulfur Bacteria Under Dark Conditions

Purple nonsulfur photosynthetic bacteria were cultured anaerobically in the absence of light by a modification of the Hungate technique. Growth was slow and resembled that of fastidious anaerobes; on yeast extract-peptone-agar medium, each cell produced about 16 descendants in 15 to 20 days. Growth was stimulated by addition of ethyl alcohol, acetate and H₂, or pyruvate and H₂. Cells grown in the presence of pyruvate and H₂ produced acetate and CO₂; each cell produced approximately 10 descendants in 24 hr under anaerobic, dark conditions. Spectrophotometric evidence obtained from cells which were the product of five generations suggests no difference between the bacteriochlorophyll and carotenoids synthesized by cells grown anaerobically under dark or light conditions. Likewise, the ultrastructure of the photosynthetic apparatus in cells grown anaerobically in the dark and in the light appears similar.     

Hibiscus sabdariffa Extract Inhibits Adhesion,Biofilm Initiation and Formation in Candida albicans

Indian Journal of Microbiology - Microbial biofilms act as reservoirs for pathogenic sessile microbes which reside inside the three dimensional matrix of the biofilm, and are thus protected against...     

Biofilm Formation by Cryptococcus neoformans Under Distinct Environmental Conditions

Cryptococcus neoformans is an opportunistic fungal pathogen with a propensity to infect the central nervous system of immune compromised individuals causing life-threatening meningoencephalitis. Cryptococcal biofilms have been described as a protective niche against microbial predators in nature and shown to enhance resistance against antifungal agents and specific mediators of host immune responses. Based on the potential importance of cryptococcal biofilms to its survival in the human host and in nature, these studies were designed to investigate those factors that mediate biofilm formation by C. neoformans. We observed that C. neoformans preferentially grew as planktonic cells when cultured under specific conditions designed to mimic growth within host tissues (37°C, neutral pH, and ~5% CO₂) or phagocytes (37°C, acidic pH, and ~5% CO₂) and as biofilms when cultured under conditions such as those encountered in the external environment (25–37°C, neutral pH, and ambient CO₂). Altogether, our studies suggest that conditions similar to those observed in its natural habitat may be conducive to biofilm formation by C. neoformans.     

MPP+ Toxicity in E. Coli Under Aerobic and Anaerobic Conditions

MPP⁺ and paraquat (PQ⁺²) are two structurally analogous and highly toxic pyridinium compounds. The mechanism of PQ⁺² toxicity is best understood in the bacterial model system. While numerous studies, in a variety of systems, have indicated the causative role of free radicals and other oxygen-derived active species in PQ⁺² toxicity, this question is yet unresolved in the case of MPP'. In this study we have used the E. coli model and have demonstrated that MPP⁺ is toxic to bacterial cells in a dose and time dependent modes. Additionally, it is shown that only in the presence of molecular oxygen, bacterial inactivation occurred. The protective effects of the chemical scavenger -mannitol -and of histidine are presented. These results are in complete accord with a free radical mechanism for MPP⁺ toxicity.