Cryptococcus neoformans Biofilm Formation Depends on Surface Support and Carbon Source and Reduces Fungal Cell Susceptibility to Heat, Cold, and UV Light

ABSTRACT The fungus Cryptococcus neoformans possesses a polysaccharide capsule and can form biofilms on medical devices. We describe the characteristics of C. neoformans biofilm development using a microtiter plate model, microscopic examinations, and a colorimetric 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay to observe the metabolic activity of cryptococci within a biofilm. A strong correlation between XTT and CFU assays was demonstrated. Chemical analysis of the exopolymeric material revealed sugar composition consisting predominantly of xylose, mannose, and glucose, indicating the presence of other polysaccharides in addition to glucurunoxylomannan. Biofilm formation was affected by surface support differences, conditioning films on the surface, characteristics of the medium, and properties of the microbial cell. A specific antibody to the capsular polysaccharide of this fungus was used to stain the extracellular polysaccharide matrix of the fungal biofilms using light and confocal microscopy. Additionally, the susceptibility of C. neoformans biofilms and planktonic cells to environmental stress was investigated using XTT reduction and CFU assays. Biofilms were less susceptible to heat, cold, and UV light exposition than their planktonic counterparts. Our findings demonstrate that fungal biofilm formation is dependent on support surface characteristics and that growth in the biofilm state makes fungal cells less susceptible to potential environmental stresses.

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Susceptibility of Cryptococcus neoformans Biofilms to Antifungal Agents In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.3.1021-1033.2006 ◽

2006 ◽

Vol 50 (3) ◽

pp. 1021-1033 ◽

Cited By ~ 109

Author(s):

Luis R. Martinez ◽

Arturo Casadevall

Keyword(s):

Confocal Microscopy ◽

Cryptococcus Neoformans ◽

Amphotericin B ◽

Biofilm Formation ◽

Antifungal Agents ◽

Capsular Polysaccharide ◽

Antifungal Drugs ◽

Enzyme Linked Immunosorbent Assay ◽

Sequence Of Events ◽

Biofilm Development

ABSTRACT Microbial biofilms contribute to virulence and resistance to antibiotics by shielding microbial cells from host defenses and antimicrobial drugs, respectively. Cryptococcus neoformans was demonstrated to form biofilms in polystyrene microtiter plates. The numbers of CFU of disaggregated biofilms, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide reduction, and light and confocal microscopy were used to measure the fungal mass, the metabolic activity, and the appearance of C. neoformans biofilms, respectively. Biofilm development by C. neoformans followed a standard sequence of events: fungal surface attachment, microcolony formation, and matrix production. The susceptibilities of C. neoformans cells of the biofilm and planktonic phenotypes to four antifungal agents were examined. The exposure of C. neoformans cells or preformed cryptococcal biofilms to fluconazole or voriconazole did not result in yeast growth inhibition and did not affect the metabolic activities of the biofilms, respectively. In contrast, both C. neoformans cells and preformed biofilms were susceptible to amphotericin B and caspofungin. However, C. neoformans biofilms were significantly more resistant to amphotericin B and caspofungin than planktonic cells, and their susceptibilities to these drugs were further reduced if cryptococcal cells contained melanin. A spot enzyme-linked immunosorbent assay and light and confocal microscopy were used to investigate how antifungal drugs affected C. neoformans biofilm formation. The mechanism by which amphotericin B and caspofungin interfered with C. neoformans biofilm formation involved capsular polysaccharide release and adherence. Our results suggest that biofilm formation may diminish the efficacies of some antifungal drugs during cryptococcal infection.

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Early Biofilm Formation on UV Light Activated Nanoporous TiO2SurfacesIn Vivo

International Journal of Biomaterials ◽

10.1155/2018/7275617 ◽

2018 ◽

Vol 2018 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Nagat Areid ◽

Eva Söderling ◽

Johanna Tanner ◽

Ilkka Kangasniemi ◽

Timo O. Närhi

Keyword(s):

Titanium Alloy ◽

Biofilm Formation ◽

Uv Light ◽

Antibacterial Properties ◽

Mutans Streptococci ◽

Human Gingival Fibroblast ◽

Gingival Fibroblast ◽

Biofilm Development

Purpose. To explore earlyS. mutansbiofilm formation on hydrothermally induced nanoporous TiO2surfacesin vivoand to examine the effect of UV light activation on the biofilm development.Materials and Methods. Ti-6Al-4V titanium alloy discs (n = 40) were divided into four groups with different surface treatments: noncoated titanium alloy (NC); UV treated noncoated titanium alloy (UVNC); hydrothermally induced TiO2coating (HT); and UV treated titanium alloy with hydrothermally induced TiO2coating (UVHT).In vivoplaque formation was studied in 10 healthy, nonsmoking adult volunteers. Titanium discs were randomly distributed among the maxillary first and second molars. UV treatment was administered for 60 min immediately before attaching the discs in subjects’ molars. Plaque samples were collected 24h after the attachment of the specimens. Mutans streptococci (MS), non-mutans streptococci, and total facultative bacteria were cultured, and colonies were counted.Results. The plaque samples of NC (NC + UVNC) surfaces showed over 2 times more oftenS. mutanswhen compared to TiO2surfaces (HT + UVHT), with the number of colonized surfaces equal to 7 and 3, respectively.Conclusion. Thisin vivostudy suggested that HT TiO2surfaces, which we earlier showed to improve blood coagulation and encourage human gingival fibroblast attachmentin vitro, do not enhance salivary microbial (mostly mutans streptococci) adhesion and initial biofilm formation when compared with noncoated titanium alloy. UV light treatment provided Ti-6Al-4V surfaces with antibacterial properties and showed a trend towards less biofilm formation when compared with non-UV treated titanium surfaces.

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EDTA Inhibits Biofilm Formation, Extracellular Vesicular Secretion, and Shedding of the Capsular Polysaccharide Glucuronoxylomannan by Cryptococcus neoformans

Applied and Environmental Microbiology ◽

10.1128/aem.01953-12 ◽

2012 ◽

Vol 78 (22) ◽

pp. 7977-7984 ◽

Cited By ~ 44

Author(s):

Emma J. Robertson ◽

Julie M. Wolf ◽

Arturo Casadevall

Keyword(s):

Cryptococcus Neoformans ◽

Biofilm Formation ◽

Capsular Polysaccharide ◽

Inhibitory Effect ◽

Antifungal Drugs ◽

Biofilm Growth ◽

Content Type ◽

Sublethal Concentrations ◽

Vesicular Secretion

ABSTRACTThe fungal pathogenCryptococcus neoformanscan grow as a biofilm on a range of synthetic and prosthetic materials. Cryptococcal biofilm formation can complicate the placement of shunts used to relieve increased intracranial pressure in cryptococcal meningitis and can serve as a nidus for chronic infection. Biofilms are generally advantageous to pathogensin vivo, as they can confer resistance to antimicrobial compounds, including fluconazole and voriconazole in the case ofC. neoformans. EDTA can inhibit biofilm formation by several microbes and enhances the susceptibility of biofilms to antifungal drugs. In this study, we evaluated the effect of sublethal concentrations of EDTA on the growth of cryptococcal biofilms. EDTA inhibited biofilm growth byC. neoformans, and the inhibition could be reversed by the addition of magnesium or calcium, implying that the inhibitory effect was by divalent cation starvation. EDTA also reduced the amount of the capsular polysaccharide glucuronoxylomannan shed into the biofilm matrix and decreased vesicular secretion from the cell, thus providing a potential mechanism for the inhibitory effect of this cation-chelating compound. Our data imply that the growth ofC. neoformansbiofilms requires the presence of divalent metals in the growth medium and suggest that cations are required for the export of materials needed for biofilm formation, possibly including extracellular vesicles.

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Genome-Wide Transposon Mutagenesis Reveals a Role for pO157 Genes in Biofilm Development in Escherichia coli O157:H7 EDL933

Infection and Immunity ◽

10.1128/iai.00156-10 ◽

2010 ◽

Vol 78 (6) ◽

pp. 2377-2384 ◽

Cited By ~ 49

Author(s):

Supraja Puttamreddy ◽

Nancy A. Cornick ◽

F. Chris Minion

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Microtiter Plate ◽

Virulence Plasmid ◽

Microtiter Plate Assay ◽

Severe Diarrhea ◽

Wide Range ◽

Intergenic Regions ◽

Biofilm Development

ABSTRACT Enterohemorrhagic Escherichia coli O157:H7, a world-wide human food-borne pathogen, causes mild to severe diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. The ability of this pathogen to persist in the environment contributes to its dissemination to a wide range of foods and food processing surfaces. Biofilms are thought to be involved in persistence, but the process of biofilm formation is complex and poorly understood in E. coli O157:H7. To better understand the genetics of this process, a mini-Tn5 transposon insertion library was constructed in strain EDL933 and screened for biofilm-negative mutants using a microtiter plate assay. Ninety-five of 11,000 independent insertions (0.86%) were biofilm negative, and transposon insertions were located in 51 distinct genes/intergenic regions that must be involved either directly or indirectly in biofilm formation. All of the 51 biofilm-negative mutants showed reduced biofilm formation on both hydrophilic and hydrophobic surfaces. Thirty-six genes were unique to this study, including genes on the virulence plasmid pO157. The type V secreted autotransporter serine protease EspP and the enterohemolysin translocator EhxD were found to be directly involved in biofilm formation. In addition, EhxD and EspP were also important for adherence to T84 intestinal epithelial cells, suggesting a role for these genes in tissue interactions in vivo.

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Specific Antibody Can Prevent Fungal Biofilm Formation and This Effect Correlates with Protective Efficacy

Infection and Immunity ◽

10.1128/iai.73.10.6350-6362.2005 ◽

2005 ◽

Vol 73 (10) ◽

pp. 6350-6362 ◽

Cited By ~ 98

Author(s):

Luis R. Martinez ◽

Arturo Casadevall

Keyword(s):

Biofilm Formation ◽

Capsular Polysaccharide ◽

Protective Efficacy ◽

Pathogenic Fungus ◽

Medical Problems ◽

Fungal Cell ◽

Antimicrobial Drug Resistance ◽

Prosthetic Devices ◽

Polysaccharide Capsule

ABSTRACT One of the most troublesome medical problems today is infection of prosthetic devices with organisms that form polysaccharide biofilms. This combined with increasing antimicrobial drug resistance is making many infectious diseases incurable. Cryptococcus neoformans is a human-pathogenic fungus that has a polysaccharide capsule and can form biofilms in prosthetic medical devices. We developed a system to study cryptococcal biofilm formation in vitro and studied the effect of antibody to the C. neoformans capsular polysaccharide on this process. C. neoformans biofilm formation was dependent on the presence of a polysaccharide capsule and correlated with the ability of capsular polysaccharide to bind the polystyrene solid support. Protective antibodies prevented biofilm formation whereas nonprotective antibodies were not effective. The mechanism of antibody action involved interference with capsular polysaccharide release from the fungal cell. In contrast, lactoferrin, an effector molecule of innate immune mechanisms, was unable to prevent fungal biofilm formation despite its efficacy against bacterial biofilms. Our results suggest a new role of adaptive humoral immunity whereby some antibodies can inhibit biofilm formation by encapsulated organisms. Vaccines that elicit antibody responses to capsular antigens and/or passive transfer of antibodies to microbial polysaccharides may be useful in preventing biofilm formation.

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Enhanced Biofilm Formation and Loss of Capsule Synthesis: Deletion of a Putative Glycosyltransferase in Porphyromonas gingivalis

Journal of Bacteriology ◽

10.1128/jb.01685-05 ◽

2006 ◽

Vol 188 (15) ◽

pp. 5510-5523 ◽

Cited By ~ 76

Author(s):

Mary E. Davey ◽

Margaret J. Duncan

Keyword(s):

Porphyromonas Gingivalis ◽

Biofilm Formation ◽

Capsular Polysaccharide ◽

Sequence Similarity ◽

Opportunistic Pathogen ◽

Insertion Mutant ◽

Wild Type ◽

High Sequence Similarity ◽

Biofilm Development

ABSTRACT Periodontitis is a biofilm-mediated disease. Porphyromonas gingivalis is an obligate anaerobe consistently associated with severe manifestations of this disease. As an opportunistic pathogen, the ability to proliferate within and disseminate from subgingival biofilm (plaque) is central to its virulence. Here, we report the isolation of a P. gingivalis transposon insertion mutant altered in biofilm development and the reconstruction and characterization of this mutation in three different wild-type strains. The mutation responsible for the altered biofilm phenotype was in a gene with high sequence similarity (∼61%) to a glycosyltransferase gene. The gene is located in a region of the chromosome that includes up to 16 genes predicted to be involved in the synthesis and transport of capsular polysaccharide. The phenotype of the reconstructed mutation in all three wild-type backgrounds is that of enhanced biofilm formation. In addition, in strain W83, a strain that is encapsulated, the glycosyltransferase mutation resulted in a loss of capsule. Further experiments showed that the W83 mutant strain was more hydrophobic and exhibited increased autoaggregation. Our results indicate that we have identified a gene involved in capsular-polysaccharide synthesis in P. gingivalis and that the production of capsule prevented attachment and the initiation of in vitro biofilm formation on polystyrene microtiter plates.

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Methamphetamine Enhances Cryptococcus neoformans Pulmonary Infection and Dissemination to the Brain

mBio ◽

10.1128/mbio.00400-13 ◽

2013 ◽

Vol 4 (4) ◽

Cited By ~ 15

Author(s):

Dhavan Patel ◽

Gunjan M. Desai ◽

Susana Frases ◽

Radames J. B. Cordero ◽

Carlos M. DeLeon-Rodriguez ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Respiratory Tract ◽

Cryptococcus Neoformans ◽

Biofilm Formation ◽

Pulmonary Infection ◽

Capsular Polysaccharide ◽

Content Type ◽

The Impact ◽

The Brain

ABSTRACTMethamphetamine (METH) is a major addictive drug of abuse in the United States and worldwide, and its use is linked to HIV acquisition. The encapsulated fungusCryptococcus neoformansis the most common cause of fungal meningitis in patients with AIDS. In addition to functioning as a central nervous system stimulant, METH has diverse effects on host immunity. Using a systemic mouse model of infection andin vitroassays in order to critically assess the impact of METH onC. neoformanspathogenesis, we demonstrate that METH stimulates fungal adhesion, glucuronoxylomannan (GXM) release, and biofilm formation in the lungs. Interestingly, structural analysis of the capsular polysaccharide of METH-exposed cryptococci revealed that METH alters the carbohydrate composition of this virulence factor, an event of adaptation to external stimuli that can be advantageous to the fungus during pathogenesis. Additionally, we show that METH promotesC. neoformansdissemination from the respiratory tract into the brain parenchyma. Our findings provide novel evidence of the impact of METH abuse on host homeostasis and increased permissiveness to opportunistic microorganisms.IMPORTANCEMethamphetamine (METH) is a major health threat to our society, as it adversely changes people’s behavior, as well as increases the risk for the acquisition of diverse infectious diseases, particularly those that enter through the respiratory tract or skin. This report investigates the effects of METH use on pulmonary infection by the AIDS-related fungusCryptococcus neoformans. This drug of abuse stimulates colonization and biofilm formation in the lungs, followed by dissemination of the fungus to the central nervous system. Notably,C. neoformansmodifies its capsular polysaccharide after METH exposure, highlighting the fungus’s ability to adapt to environmental stimuli, a possible explanation for its pathogenesis. The findings may translate into new knowledge and development of therapeutic and public health strategies to deal with the devastating complications of METH abuse.

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Disarming Fungal Pathogens:Bacillus safensisInhibits Virulence Factor Production and Biofilm Formation byCryptococcus neoformansandCandida albicans

mBio ◽

10.1128/mbio.01537-17 ◽

2017 ◽

Vol 8 (5) ◽

Cited By ~ 24

Author(s):

François L. Mayer ◽

James W. Kronstad

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Cryptococcus Neoformans ◽

Virulence Factors ◽

Virulence Factor ◽

Biofilm Formation ◽

Fungal Pathogens ◽

Soil Bacterium ◽

Fungal Cell ◽

Virulence Factor Production

ABSTRACTBacteria interact with each other in nature and often compete for limited nutrient and space resources. However, it is largely unknown whether and how bacteria also interact with human fungal pathogens naturally found in the environment. Here, we identified a soil bacterium,Bacillus safensis, which potently blocked several keyCryptococcus neoformansvirulence factors, including formation of the antioxidant pigment melanin and production of the antiphagocytic polysaccharide capsule. The bacterium also inhibitedde novocryptococcal biofilm formation but had only modest inhibitory effects on already formed biofilms or planktonic cell growth. The inhibition of fungal melanization was dependent on direct cell contact and live bacteria.B. safensisalso had anti-virulence factor activity against another major human-associated fungal pathogen,Candida albicans. Specifically, dual-species interaction studies revealed that the bacterium strongly inhibitedC. albicansfilamentation and biofilm formation. In particular,B. safensisphysically attached to and degraded candidal filaments. Through genetic and phenotypic analyses, we demonstrated that bacterial chitinase activity against fungal cell wall chitin is a factor contributing to the antipathogen effect ofB. safensis.IMPORTANCEPathogenic fungi are estimated to contribute to as many human deaths as tuberculosis or malaria. Two of the most common fungal pathogens,Cryptococcus neoformansandCandida albicans, account for up to 1.4 million infections per year with very high mortality rates. Few antifungal drugs are available for treatment, and development of novel therapies is complicated by the need for pathogen-specific targets. Therefore, there is an urgent need to identify novel drug targets and new drugs. Pathogens use virulence factors during infection, and it has recently been proposed that targeting these factors instead of the pathogen itself may represent a new approach to develop antimicrobials. Here, we identified a soil bacterium that specifically blocked virulence factor production and biofilm formation byC. neoformansandC. albicans. We demonstrate that the bacterial antipathogen mechanism is based in part on targeting the fungal cell wall, a structure not found in human cells.

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Cyclic-di-GMP Regulates Extracellular Polysaccharide Production, Biofilm Formation, and Rugose Colony Development by Vibrio vulnificus

Applied and Environmental Microbiology ◽

10.1128/aem.00176-08 ◽

2008 ◽

Vol 74 (13) ◽

pp. 4199-4209 ◽

Cited By ~ 61

Author(s):

Alina Nakhamchik ◽

Caroline Wilde ◽

Dean A. Rowe-Magnus

Keyword(s):

Biofilm Formation ◽

Capsular Polysaccharide ◽

Vibrio Vulnificus ◽

Genomic Library ◽

Extracellular Polysaccharide ◽

Invasive Disease ◽

Colony Development ◽

Independent Manner ◽

Disease Agent ◽

And Control

ABSTRACT Vibrio vulnificus is a human and animal pathogen that carries the highest death rate of any food-borne disease agent. It colonizes shellfish and forms biofilms on the surfaces of plankton, algae, fish, and eels. Greater understanding of biofilm formation by the organism could provide insight into approaches to decrease its load in filter feeders and on biotic surfaces and control the occurrence of invasive disease. The capsular polysaccharide (CPS), although essential for virulence, is not required for biofilm formation under the conditions used here. In other bacteria, increased biofilm formation often correlates with increased exopolysaccharide (EPS) production. We exploited the translucent phenotype of acapsular mutants to screen a V. vulnificus genomic library and identify genes that imparted an opaque phenotype to both CPS biosynthesis and transport mutants. One of these encoded a diguanylate cyclase (DGC), an enzyme that synthesizes bis-(3′-5′)-cyclic-di-GMP (c-di-GMP). This prompted us to use this DGC, DcpA, to examine the effect of elevated c-di-GMP levels on several developmental pathways in V. vulnificus. Increased c-di-GMP levels induced the production of an EPS that was distinct from the CPS and dramatically enhanced biofilm formation and rugosity in a CPS-independent manner. However, the EPS could not compensate for the loss of CPS production that is required for virulence. In contrast to V. cholerae, motility and virulence appeared unaffected by elevated levels of c-di-GMP.

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Anthranilate Deteriorates the Structure of Pseudomonas aeruginosa Biofilms and Antagonizes the Biofilm-Enhancing Indole Effect

Applied and Environmental Microbiology ◽

10.1128/aem.03551-14 ◽

2015 ◽

Vol 81 (7) ◽

pp. 2328-2338 ◽

Cited By ~ 23

Author(s):

Soo-Kyoung Kim ◽

Ha-Young Park ◽

Joon-Hee Lee

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Shear Force ◽

Degradation Products ◽

Early Stage ◽

Bacterial Species ◽

Extracellular Polysaccharide ◽

Content Type ◽

Tryptophan Degradation ◽

Biofilm Development

ABSTRACTAnthranilate and indole are alternative degradation products of tryptophan, depending on the bacterial species. While indole enhances the biofilm formation ofPseudomonas aeruginosa, we found that anthranilate, the tryptophan degradation product ofP. aeruginosa, had an opposite effect onP. aeruginosabiofilm formation, in which anthranilate deteriorated the mushroom structure of biofilm. The anthranilate effect on biofilm formation was differentially exerted depending on the developmental stage and the presence of shear force. Anthranilate slightly accelerated the initial attachment ofP. aeruginosaat the early stage of biofilm development and appeared to build more biofilm without shear force. But anthranilate weakened the biofilm structure in the late stage, deteriorating the mushroom structure of biofilms with shear force to make a flat biofilm. To investigate the interplay of anthranilate with indole in biofilm formation, biofilms were cotreated with anthranilate and indole, and the results showed that anthranilate antagonized the biofilm-enhancing effect of indole. Anthranilate was able to deteriorate the preformed biofilm. The effect of anthranilate and indole on biofilm formation was quorum sensing independent. AntR, a regulator of anthranilate-degrading metabolism was synergistically activated by cotreatment with anthranilate and indole, suggesting that indole might enhance biofilm formation by facilitating the degradation of anthranilate. Anthranilate slightly but significantly affected the cyclic diguaniylate (c-di-GMP) level and transcription of major extracellular polysaccharide (Psl, Pel, and alginate) operons. These results suggest that anthranilate may be a promising antibiofilm agent and antagonize the effect of indole onP. aeruginosabiofilm formation.

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