Publications by authors named "Bastiaan P Krom"

72 Publications

Immunoediting role for major vault protein in apoptotic signaling induced by bacterial -acyl homoserine lactones.

Proc Natl Acad Sci U S A 2021 Mar;118(12)

Department of Chemistry and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be'er Sheva 8410501, Israel;

The major vault protein (MVP) mediates diverse cellular responses, including cancer cell resistance to chemotherapy and protection against inflammatory responses to Here, we report the use of photoactive probes to identify MVP as a target of the -(3-oxo-dodecanoyl) homoserine lactone (C12), a quorum sensing signal of certain proteobacteria including A treatment of normal and cancer cells with C12 or other -acyl homoserine lactones (AHLs) results in rapid translocation of MVP into lipid raft (LR) membrane fractions. Like AHLs, inflammatory stimuli also induce LR-localization of MVP, but the C12 stimulation reprograms (functionalizes) bioactivity of the plasma membrane by recruiting death receptors, their apoptotic adaptors, and caspase-8 into LR. These functionalized membranes control AHL-induced signaling processes, in that MVP adjusts the protein kinase p38 pathway to attenuate programmed cell death. Since MVP is the structural core of large particles termed vaults, our findings suggest a mechanism in which MVP vaults act as sentinels that fine-tune inflammation-activated processes such as apoptotic signaling mediated by immunosurveillance cytokines including tumor necrosis factor-related apoptosis inducing ligand (TRAIL).
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http://dx.doi.org/10.1073/pnas.2012529118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000436PMC
March 2021

The novel endolysin XZ.700 effectively treats MRSA biofilms in two biofilm models without showing toxicity on human bone cells .

Biofouling 2021 02 21;37(2):184-193. Epub 2021 Feb 21.

Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.

In this study the effect of XZ.700, a new endolysin, on methicillin resistant (MRSA) biofilms grown on titanium was evaluated. Biofilms of USA300 were grown statically and under flow, and treatment with XZ.700 was compared with povidone-iodine (PVP-I) and gentamicin. To evaluate the cytotoxic effects of XZ.700 and derived biofilm lysates, human osteocyte-like cells were exposed to biofilm supernatants, and metabolism and proliferation were quantified. XZ.700 showed a significant, concentration dependent reduction in biofilm viability, compared with carrier controls. Metabolism and proliferation of human osteocyte-like cells were not affected by XZ.700 or lysates, unlike PVP-I and gentamicin lysates which significantly inhibited proliferation. Using time-lapse microscopy, rapid biofilm killing and removal was observed for XZ.700. In comparison, PVP-I and gentamicin showed slower biofilm killing, with no apparent biofilm removal. In conclusion, XZ.700 reduced MRSA biofilms, especially under flow condition, without toxicity for surrounding bone cells.
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http://dx.doi.org/10.1080/08927014.2021.1887151DOI Listing
February 2021

Adhesion of to During Co-Infection Promotes Bacterial Dissemination Through the Host Immune Response.

Front Cell Infect Microbiol 2020 2;10:624839. Epub 2021 Feb 2.

Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Department of Biology, KU Leuven, Leuven, Belgium.

Interspecies interactions greatly influence the virulence, drug tolerance and ultimately the outcome of polymicrobial biofilm infections. A synergistic interaction is observed between the fungus and the bacterium . These species are both normal commensals of most healthy humans and co-exist in several niches of the host. However, under certain circumstances, they can cause hospital-acquired infections with high morbidity and mortality rates. Using a mouse model of oral co-infection, we previously showed that an oral infection with predisposes to a secondary systemic infection with . Here, we unraveled this intriguing mechanism of bacterial dissemination. Using static and dynamic adhesion assays in combination with single-cell force spectroscopy, we identified Als1 and Als3 adhesins as the molecular players involved in the interaction with and in subsequent bacterial dissemination. Remarkably, we identified the host immune response as a key element required for bacterial dissemination. We found that the level of immunosuppression of the host plays a critical yet paradoxical role in this process. In addition, secretion of candidalysin, the peptide responsible for immune activation and cell damage, is required for colonization and subsequent bacterial dissemination. The physical interaction with enhances bacterial uptake by phagocytic immune cells, thereby enabling an opportunity to disseminate.
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http://dx.doi.org/10.3389/fcimb.2020.624839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884861PMC
June 2021

DNase-mediated eDNA removal enhances D-LL-31 activity against biofilms of bacteria isolated from chronic rhinosinusitis patients.

Biofouling 2020 10 9;36(9):1117-1128. Epub 2020 Dec 9.

Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.

Chronic rhinosinusitis (CRS) is a chronic infection of the nasal cavity and paranasal sinuses associated with the presence of a microbial biofilm. Extracellular DNA (eDNA) is an important component of the biofilm matrix. Antimicrobial peptides (AMPs) are natural peptides with the ability to kill microorganisms. D-LL-31 is a synthetic variant of the AMP cathelicidin with increased resistance to proteolytic breakdown. In this study it is shown for 3 clinical CRS isolates that treatment of 24 h biofilms with DNase I enhanced the antimicrobial activity of D-LL-31. Conversely, co-incubation of D-LL-31 at the IC value with exogenous DNA resulted in reduced antimicrobial activity. DNase I alone did not show antimicrobial activity against the isolates tested but caused dispersal of an established biofilm. Hence, the presence of eDNA in the biofilm matrix reduced AMP-mediated killing. These results suggest that combination therapy with proteolysis resistant AMP D-LL-31 and DNase could be considered for effective treatment of CRS.
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http://dx.doi.org/10.1080/08927014.2020.1857741DOI Listing
October 2020

Of fungi and men: role of fungi in pancreatic cancer carcinogenesis.

Ann Transl Med 2020 Oct;8(19):1257

Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Amsterdam, The Netherlands.

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http://dx.doi.org/10.21037/atm-20-2723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7607088PMC
October 2020

Short-Chain -Acylhomoserine Lactone Quorum-Sensing Molecules Promote Periodontal Pathogens in Oral Biofilms.

Appl Environ Microbiol 2020 01 21;86(3). Epub 2020 Jan 21.

Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam, The Netherlands

Acylhomoserine lactones (AHLs), the quorum-sensing (QS) signals produced by a range of Gram-negative bacteria, are involved in biofilm formation in many pathogenic and environmental bacteria. Nevertheless, the current paradigm excludes a role of AHLs in dental plaque formation, while other QS signals, such as AI-2 and autoinducer peptides, have been demonstrated to play an important role in biofilm formation and virulence-related gene expression in oral pathogens. In the present work, we have explored the effect of externally added AHLs on oral biofilm models for commensal, cariogenic, and periodontal dental plaque. While little effect on bacterial growth was observed, some AHLs specifically affected the lactic acid production and protease activity of the biofilms. Most importantly, the analysis of bacterial diversity in the biofilms showed that the addition of C-homoserine lactone (C-HSL) results in a shift toward a periodontal bacterial composition profile by increasing the relative presence of the orange-complex bacteria and These results point to a relevant role of AHL-mediated QS in dental plaque formation and might be involved in the development of dysbiosis, the mechanism of which should be further investigated. This finding potentially opens new opportunities for the prevention or treatment of the periodontal disease. Dental plaque is omnipresent in healthy oral cavities and part of our commensal microbial colonization. At the same time, dental plaque is the cause of the most common human diseases, caries and gum disease. Dental plaque consists of billions of microbes attached to the surface of your teeth. Communication among these microbes is pivotal for development of these complex communities yet poorly studied in dental plaque. In the present study, we show that a specific communication molecule induces changes within the community related to the development of gum disease. This finding suggests that interfering with microbial communication may represent an interesting novel strategy to prevent gum disease that should be further investigated.
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http://dx.doi.org/10.1128/AEM.01941-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6974637PMC
January 2020

The Host Immune System Facilitates Disseminated Staphylococcus aureus Disease Due to Phagocytic Attraction to Candida albicans during Coinfection: a Case of Bait and Switch.

Infect Immun 2019 11 18;87(11). Epub 2019 Oct 18.

Department of Microbial Pathogenesis, School of Dentistry, University of Maryland-Baltimore, Baltimore, Maryland, USA.

Invasive infections account for 15 to 50% of fatal bloodstream infections annually. These disseminated infections often arise without a defined portal of entry into the host but cause high rates of mortality. The fungus and the Gram-positive bacterium can form polymicrobial biofilms on epithelial tissue, facilitated by the adhesin encoded by While a bacterium-fungus interaction is required for systemic infection, the mechanism by which bacteria disseminate from the epithelium to internal organs is unclear. In this study, we show that highly immunogenic hyphae attract phagocytic cells, which rapidly engulf adherent and subsequently migrate to cervical lymph nodes. Following -loaded phagocyte translocation from the mucosal surface, produces systemic disease with accompanying morbidity and mortality. Our results suggest a novel role for the host in facilitating a bacterium-fungus infectious synergy, leading to disseminated staphylococcal disease.
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http://dx.doi.org/10.1128/IAI.00137-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6803350PMC
November 2019

enhances initial biofilm growth of under aerobic conditions.

Biofouling 2019 03 14;35(3):350-360. Epub 2019 May 14.

a Laboratoire Ecologie Biologie des Interactions - UMR CNRS 7267 , Université de Poitiers , Poitiers , France.

and are opportunistic pathogens that co-colonize the human body. They are involved in biofilm-related infections of implanted medical devices. The objective of this study was to evaluate the ability of these species to interact and form polymicrobial biofilms. SEM imaging and adhesion assays showed that adhesion to did not have a preference for a specific morphological state of ; bacteria adhered to both hyphal and yeast forms of . did not influence growth of under anaerobic growth conditions, however under aerobic growth condition, enhanced early biofilm formation. This favorable impact of was not mediated by secreted compounds accumulating in the medium, but required the presence of metabolically active . The ability of these microorganisms to interact together could modulate the physiopathology of infections.
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http://dx.doi.org/10.1080/08927014.2019.1608966DOI Listing
March 2019

Review: modulation of the oral microbiome by the host to promote ecological balance.

Odontology 2019 Oct 4;107(4):437-448. Epub 2019 Feb 4.

Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, G. Mahlerlaan 3004, 1081 LA, Amsterdam, The Netherlands.

The indivisible relationship between the human host and its oral microbiome has been shaped throughout the millennia, by facing various changes that have forced the adaptation of oral microorganisms to new environmental conditions. In this constant crosstalk between the human host and its microbiome, a bidirectional relationship has been established. The microorganisms provide the host with functions it cannot perform on its own and at the same time the host provides its microbes with a suitable environment for their growth and development. These host factors can positively affect the microbiome, promoting diversity and balance between different species, resulting in a state of symbiosis and absence of pathology. In contrast, other host factors can negatively influence the composition of the oral microbiome and drive the interaction towards a dysbiotic state, where the balance tilts towards a harmful relationship between the host and its microbiome. The aim of this review is to describe the role host factors play in cultivating and maintaining a healthy oral ecology and discuss mechanisms that can prevent its drift towards dysbiosis.
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http://dx.doi.org/10.1007/s10266-019-00413-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6732124PMC
October 2019

Impact of nutritional stress on drug susceptibility and biofilm structures of Burkholderia pseudomallei and Burkholderia thailandensis grown in static and microfluidic systems.

PLoS One 2018 26;13(3):e0194946. Epub 2018 Mar 26.

Biofilm Research Group, Faculty of Dentistry, Khon Kaen University, Khon Kaen, Thailand.

Burkholderia pseudomallei is the causative agent of melioidosis and regarded as a bioterrorism threat. It can adapt to the nutrient-limited environment as the bacteria can survive in triple distilled water for 16 years. Moreover, B. pseudomallei exhibits intrinsic resistance to diverse groups of antibiotics in particular while growing in biofilms. Recently, nutrient-limited condition influenced both biofilm formation and ceftazidime (CAZ) tolerance of B. pseudomallei were found. However, there is no information about how nutrient-limitation together with antibiotics used in melioidosis treatment affects the structure of the biofilm produced by B. pseudomallei. Moreover, no comparative study to investigate the biofilm architectures of B. pseudomallei and the related B. thailandensis under different nutrient concentrations has been reported. Therefore, this study aims to provide new information on the effects of four antibiotics used in melioidosis treatment, viz. ceftazidime (CAZ), imipenem (IMI), meropenem (MEM) and doxycycline (DOX) on biofilm architecture of B. pseudomallei and B. thailandensis with different nutrient concentrations under static and flow conditions using confocal laser scanning microscopy. Impact of nutritional stress on drug susceptibility of B. pseudomallei and B. thailandensis grown planktonically or as biofilm was also evaluated. The findings of this study indicate that nutrient-limited environment enhanced survival of B. pseudomallei in biofilm after exposure to the tested antibiotics. The shedding planktonic B. pseudomallei and B. thailandensis were also found to have increased CAZ tolerance in nutrient-limited environment. However, killing activities of MEM and IMI were stronger than CAZ and DOX on B. pseudomallei and B. thailandensis both in planktonic cells and in 2-day old biofilm. In addition, MEM and IMI were able to inhibit B. pseudomallei and B. thailandensis biofilm formation to a larger extend compared to CAZ and DOX. Differences in biofilm architecture were observed for biofilms grown under static and flow conditions. Under static conditions, biofilms grown in full strength modified Vogel and Bonner's medium (MVBM) showed honeycomb-like architecture while a knitted-like structure was observed under limited nutrient condition (0.1×MVBM). Under flow conditions, biofilms grown in MVBM showed a multilayer structure while merely dispersed bacteria were found when grown in 0.1×MVBM. Altogether, this study provides more insight on the effect of four antibiotics against B. pseudomallei and B. thailandensis in biofilm under different nutrient and flow conditions. Since biofilm formation is believed to be involved in disease relapse, MEM and IMI may be better therapeutic options than CAZ for melioidosis treatment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194946PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5868842PMC
July 2018

Bacterial-fungal interactions: ecology, mechanisms and challenges.

FEMS Microbiol Rev 2018 05;42(3):335-352

Helmholtz Centre for Environmental Research-UFZ, Department of Environmental Microbiology, Permoserstraße 15, 04318 Leipzig, Germany.

Fungi and bacteria are found living together in a wide variety of environments. Their interactions are significant drivers of many ecosystem functions and are important for the health of plants and animals. A large number of fungal and bacterial families engage in complex interactions that lead to critical behavioural shifts of the microorganisms ranging from mutualism to antagonism. The importance of bacterial-fungal interactions (BFI) in environmental science, medicine and biotechnology has led to the emergence of a dynamic and multidisciplinary research field that combines highly diverse approaches including molecular biology, genomics, geochemistry, chemical and microbial ecology, biophysics and ecological modelling. In this review, we discuss recent advances that underscore the roles of BFI across relevant habitats and ecosystems. A particular focus is placed on the understanding of BFI within complex microbial communities and in regard of the metaorganism concept. We also discuss recent discoveries that clarify the (molecular) mechanisms involved in bacterial-fungal relationships, and the contribution of new technologies to decipher generic principles of BFI in terms of physical associations and molecular dialogues. Finally, we discuss future directions for research in order to stimulate synergy within the BFI research area and to resolve outstanding questions.
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http://dx.doi.org/10.1093/femsre/fuy008DOI Listing
May 2018

Metabolic Interactions between Bacteria and Fungi in Commensal Oral Biofilms.

J Fungi (Basel) 2017 Jul 14;3(3). Epub 2017 Jul 14.

Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam (ACTA), Vrije Universiteit Amsterdam and the University of Amsterdam, Amsterdam, 1081 LA, The Netherlands.

Oral health is more than just the absence of disease. The key to oral health is a diverse microbiome in an ecological balance. The oral microbiota is one of the most complex and diverse microbial communities in the human body. To maintain oral health, balance between the human host and the intrinsic microorganisms is essential. The healthy oral cavity is represented by a great microbial diversity, including both bacteria and fungi. The bacterial microbiome is very well studied. In contrast, fungi inhabiting the oral cavity are often overlooked. All microbial species in the oral cavity form communities which establish a variety of micro-niches and inter- and intra-species interactions. These interactions can be classified into three main groups: physical, chemical and metabolic interactions. Different metabolic interactions are reviewed in this report, among which are the metabolism of sugars, carbon, lactate and oxygen. This review set out with the aim of assessing the importance of metabolic interactions between fungi and bacteria in the healthy oral cavity.
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http://dx.doi.org/10.3390/jof3030040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5715944PMC
July 2017

Phytosphingosine Prevents the Formation of Young Salivary Biofilms in vitro.

Caries Res 2018 13;52(1-2):7-13. Epub 2017 Dec 13.

Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam (ACTA), Free University of Amsterdam and University of Amsterdam, Amsterdam, The Netherlands.

Dental biofilms are formed in a multistep process that is initiated by the adhesion of oral bacteria to the dental hard surface. As dental biofilms are associated with oral diseases their control is necessary in order to maintain oral health. Recently, it was revealed that phytosphingosine (PHS)-treated hydroxyapatite discs showed anti-adhesive activity in a static in vitro biofilm model against Streptococcus mutans. The goal of the present study was to further unravel the anti-adhesive and anti-biofilm properties of PHS in both static and dynamic in vitro biofilm models against a full salivary inoculum. After 3 h under static conditions, bacterial adherence on PHS-treated cover glass slides was reduced by 60% compared to the untreated surface. After 6 and 24 h under static conditions, no significant differences in bacterial adherence were observed between PHS-treated and untreated cover glass slides. However, under dynamic conditions, i.e., the presence of shear forces, virtually no bacterial adherence was observed for up to 16 h on PHS-coated surfaces. Besides, PHS showed a strong bactericidal activity on salivary biofilms. Treatment of a 3- and 6-h statically grown biofilm resulted in a 99 and 94% reduction of viable cells, respectively, which was effectuated within minutes. In principle, these anti-adherence and anti-biofilm properties make PHS a promising candidate ingredient for use in oral care products aimed at oral microbial control.
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http://dx.doi.org/10.1159/000480712DOI Listing
April 2019

Fungal mitochondrial oxygen consumption induces the growth of strict anaerobic bacteria.

Fungal Genet Biol 2017 12 5;109:1-6. Epub 2017 Oct 5.

Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam, The Netherlands. Electronic address:

Fungi are commonly encountered as part of a healthy oral ecosystem. Candida albicans is the most often observed and investigated fungal species in the oral cavity. The role of fungi in the oral ecosystem has remained enigmatic for decades. Recently, it was shown that C. albicans, in vitro, influences the bacterial composition of young oral biofilms, indicating it possibly plays a role in increasing diversity in the oral ecosystem. C. albicans favored growth of strictly anaerobic species under aerobic culture conditions. In the present study, the role of mitochondrial respiration, as mechanism by which C. albicans modifies its environment, was investigated. Using oxygen sensors, a rapid depletion of dissolved oxygen (dO) was observed. This decrease was not C. albicans specific as several non-albicans Candida species showed similar oxygen consumption. Heat inactivation as well as addition of the specific mitochondrial respiration inhibitor Antimycin A inhibited depletion of dO. Using 16S rDNA sequencing, it is shown that mitochondrial activity, more than physical presence of C. albicans is responsible for inducing growth of strictly anaerobic oral bacteria in aerobic growth conditions. The described mechanism of dO depletion may be a general mechanism by which fungi modulate their direct environment.
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http://dx.doi.org/10.1016/j.fgb.2017.10.001DOI Listing
December 2017

Ica-status of clinical Staphylococcus epidermidis strains affects adhesion and aggregation: a thermodynamic analysis.

Antonie Van Leeuwenhoek 2017 Nov 12;110(11):1467-1474. Epub 2017 Jun 12.

Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, Amsterdam, The Netherlands.

Staphylococcus epidermidis is a major nosocomial pathogen associated with infections of indwelling medical devices. One important virulence factor of these organisms is their ability to adhere to devices and form biofilms. In this study, we evaluated the effect of the ica operon on cell surface hydrophobicity, thermodynamics of adhesion, and biofilm formation for seven S. epidermidis strains. The surface free energy parameters of the bacterial cell surface and the substratum were determined by contact angle measurement. Biofilm formation was assayed using crystal violet staining. Results showed that ica-positive strains demonstrated a higher hydrophobic characteristic than ica-negative strains, suggesting that the ica-operon seems to determine the cell surface hydrophobicity of S. epidermidis. Interaction of ica-positive strains with a tissue-culture treated polystyrene surface was energetically favourable (ΔG < 0), in contrast to ica-negative strains (ΔG > 0). The interfacial free energy of aggregation of S. epidermidis was lower for ica-positive than for ica-negative strains. Our study suggests that, in addition to biofilm formation, adhesion and aggregation of clinical S. epidermidis is stimulated in ica-positive strains by influencing the thermodynamics of interaction.
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http://dx.doi.org/10.1007/s10482-017-0899-2DOI Listing
November 2017

On the ecosystemic network of saliva in healthy young adults.

ISME J 2017 05 10;11(5):1218-1231. Epub 2017 Jan 10.

Top Institute Food and Nutrition, Wageningen, The Netherlands.

A dysbiotic state is believed to be a key factor in the onset of oral disease. Although oral diseases have been studied for decades, our understanding of oral health, the boundaries of a healthy oral ecosystem and ecological shift toward dysbiosis is still limited. Here, we present the ecobiological heterogeneity of the salivary ecosystem and relations between the salivary microbiome, salivary metabolome and host-related biochemical salivary parameters in 268 healthy adults after overnight fasting. Gender-specific differences in the microbiome and metabolome were observed and were associated with salivary pH and dietary protein intake. Our analysis grouped the individuals into five microbiome and four metabolome-based clusters that significantly related to biochemical parameters of saliva. Low salivary pH and high lysozyme activity were associated with high proportions of streptococcal phylotypes and increased membrane-lipid degradation products. Samples with high salivary pH displayed increased chitinase activity, higher abundance of Veillonella and Prevotella species and higher levels of amino acid fermentation products, suggesting proteolytic adaptation. An over-specialization toward either a proteolytic or a saccharolytic ecotype may indicate a shift toward a dysbiotic state. Their prognostic value and the degree to which these ecotypes are related to increased disease risk remains to be determined.
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http://dx.doi.org/10.1038/ismej.2016.199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5475835PMC
May 2017

Red and Green Fluorescence from Oral Biofilms.

PLoS One 2016 20;11(12):e0168428. Epub 2016 Dec 20.

Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam, the Netherlands.

Red and green autofluorescence have been observed from dental plaque after excitation by blue light. It has been suggested that this red fluorescence is related to caries and the cariogenic potential of dental plaque. Recently, it was suggested that red fluorescence may be related to gingivitis. Little is known about green fluorescence from biofilms. Therefore, we assessed the dynamics of red and green fluorescence in real-time during biofilm formation. In addition, the fluorescence patterns of biofilm formed from saliva of eight different donors are described under simulated gingivitis and caries conditions. Biofilm formation was analysed for 12 hours under flow conditions in a microfluidic BioFlux flow system with high performance microscopy using a camera to allow live cell imaging. For fluorescence images dedicated excitation and emission filters were used. Both green and red fluorescence were linearly related with the total biomass of the biofilms. All biofilms displayed to some extent green and red fluorescence, with higher red and green fluorescence intensities from biofilms grown in the presence of serum (gingivitis simulation) as compared to the sucrose grown biofilms (cariogenic simulation). Remarkably, cocci with long chain lengths, presumably streptococci, were observed in the biofilms. Green and red fluorescence were not found homogeneously distributed within the biofilms: highly fluorescent spots (both green and red) were visible throughout the biomass. An increase in red fluorescence from the in vitro biofilms appeared to be related to the clinical inflammatory response of the respective saliva donors, which was previously assessed during an in vivo period of performing no-oral hygiene. The BioFlux model proved to be a reliable model to assess biofilm fluorescence. With this model, a prediction can be made whether a patient will be prone to the development of gingivitis or caries.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0168428PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5173178PMC
June 2017

Interspecies Interactions between and .

mSphere 2016 Nov-Dec;1(6). Epub 2016 Nov 9.

Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam (ACTA), Free University of Amsterdam and the University of Amsterdam, Amsterdam, The Netherlands.

The facultative anaerobic polymorphic fungus and the strictly anaerobic Gram-positive bacterium are two opportunistic pathogens residing in the human gut. While a few studies have focused on the prevalence of in -infected patients, the nature of the interactions between these two microbes has not been studied thus far. In the current study, both chemical and physical interactions between and were investigated. In the presence of , was able to grow under aerobic, normally toxic, conditions. This phenomenon was neither linked to adherence of bacteria to hyphae nor to biofilm formation by . Conditioned medium of inhibited hyphal growth of , which is an important virulence factor of the fungus. In addition, it induced hypha-to-yeast conversion. -Cresol, a fermentation product of tyrosine produced by , also induced morphological effects and was identified as an active component of the conditioned medium. This study shows that in the presence of , can persist and grow under aerobic conditions. Furthermore, -cresol, produced by , is involved in inhibiting hypha formation of , directly affecting the biofilm formation and virulence of . This study is the first detailed characterization of the interactions between these two gut pathogens. and are two opportunistic pathogens that reside in the human gut. A few studies have focused on the prevalence of in -infected patients, but none have shown the interaction(s) that these two organisms may or may not have with each other. In this study, we used a wide range of different techniques to better understand this interaction at a macroscopic and microscopic level. We found that in the presence of , can survive under ambient aerobic conditions, which would otherwise be toxic. We also found that affects the hypha formation of , most likely through the excretion of -cresol. This ultimately leads to an inability of to form a biofilm. Our study provides new insights into interactions between and and bears relevance to both fungal and bacterial disease.
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http://dx.doi.org/10.1128/mSphere.00187-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5103046PMC
November 2016

The mycobiome of root canal infections is correlated to the bacteriome.

Clin Oral Investig 2017 Jun 22;21(5):1871-1881. Epub 2016 Oct 22.

Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, Gustav Mahlerlaan 3004, 1081, LA, Amsterdam, The Netherlands.

Objectives: Bacterial infection of the root canal system causes apical periodontitis. Less is known about the role of fungi in these infections. This study aimed to assess the fungal prevalence, abundance, and diversity of root canal infections, as well as the relation between fungi and bacteria present in different parts of the root canal.

Materials And Methods: Twenty-six teeth with primary apical periodontitis were extracted, split in apical and coronal root segments, and cryo-pulverized. Bacteriome profiles of 23 teeth were analyzed based on the V3-V4 hypervariable region of the 16S ribosomal RNA gene. Mycobiome profiles of six teeth were analyzed based on the internal transcribed spacer (ITS) 1 or ITS2 region. Samples were sequenced on the Illumina MiSeq platform.

Results: A total of 338 bacterial operational taxonomic units (OTUs), 28 ITS1 OTUs, and 24 ITS2 OTUs were identified. Candida and Malassezia were the most frequently identified fungi. No differences could be found between the bacteriome and mycobiome profiles of the apical and coronal root segments. The bacteriome of fungi-positive root segments contained more Actinomyces, Bifidobacterium, four different Lactobacillus OTUs, Propionibacterium, and Streptococcus. A Spearman correlation matrix between bacteriomes and mycobiomes identified no correlations, but separate clusters could be observed.

Conclusions: A considerable proportion of the root canal infections contain fungi, although fungal diversity is limited. However, when fungi are present, the composition of the bacteriome is clearly different.

Clinical Relevance: Interaction between bacteria and fungi in root canal infections may complicate the infection and require alternative treatment strategies.
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http://dx.doi.org/10.1007/s00784-016-1980-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5442261PMC
June 2017

A novel compound to maintain a healthy oral plaque ecology in vitro.

J Oral Microbiol 2016 29;8:32513. Epub 2016 Jul 29.

Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Amsterdam, The Netherlands.

Objective: Dental caries is caused by prolonged episodes of low pH due to acid production by oral biofilms. Bacteria within such biofilms communicate via quorum sensing (QS). QS regulates several phenotypic biofilm parameters, such as biofilm formation and the production of virulence factors. In this study, we evaluated the effect of several QS modifiers on growth and the cariogenic potential of microcosm oral biofilms.

Methods: Biofilms were inoculated with pooled saliva and cultured in the presence of sucrose for 48 and 96 h. QS modifiers (or carrier controls) were continuously present. Lactic acid accumulation capacities were compared to evaluate the cariogenic potential of the biofilms. Subsequently, biofilm growth was quantified by determining colony forming unit counts (CFUs) and their ecology by 16S rDNA-based microbiome analyses. The minimal inhibitory concentration (MIC) for several Streptococcus spp. was determined using microbroth dilution.

Results: Of the tested QS modifiers only 3-oxo-N-(2-oxocyclohexyl)dodecanamide (3-Oxo-N) completely abolished lactic acid accumulation by the biofilms without affecting biofilm growth. This compound was selected for further investigation. The active range of 3-Oxo-N was 10-100 µM. The homologous QS molecule, acyl homoserine lactone C12, did not counteract the reduction in lactic acid accumulation, suggesting a mechanism other than QS inhibition. Microbial ecology analyses showed a reduction in the relative abundance of Streptococcus spp. in favor of the relative abundance of Veillonella spp. in the 3-Oxo-N exposed biofilms. The MIC of 3-Oxo-N for several streptococcal species varied between 8 and 32 µM.

Conclusion: 3-Oxo-N changes the ecological homeostasis of in vitro dental plaque. It reduces its cariogenic potential by minimizing lactic acid accumulation. Based on our in vitro data, 3-Oxo-N represents a promising compound in maintaining a healthy, non-cariogenic, ecology in in vivo dental plaque.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967710PMC
http://dx.doi.org/10.3402/jom.v8.32513DOI Listing
August 2016

Candida albicans in Multispecies Oral Communities; A Keystone Commensal?

Adv Exp Med Biol 2016;931:13-20

Top Institute Food and Nutrition, Wageningen, The Netherlands.

The complexity of the oral cavity, in which many hundreds of microbial species interact represents a challenge for modern microbiologists. What are all these species doing there? And why do we accept so many opportunistic pathogens to be part of our health (commensal) microflora? While the role of bacteria are often being studied, the role of fungi in the interactions within the oral cavity are understudied. This is partly because fungi in the oral cavity are generally considered as pathogens and related to diseases. In this chapter we will explore mechanisms of interaction between bacteria and fungi in the oral cavity that are involved in maintenance of oral health. We will argue that fungi in general and C. albicans specifically, should be regarded a keystone commensal in the oral cavity.
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http://dx.doi.org/10.1007/5584_2016_5DOI Listing
June 2017

Candida albicans in oral biofilms could prevent caries.

Pathog Dis 2016 07 28;74(5). Epub 2016 Apr 28.

Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, Amsterdam, The Netherlands

Streptococcus mutans is a Gram-positive bacterium involved in development to caries, the most common infectious disease of our time. Streptococcus mutans interacts with other microbes, like the fungus Candida albicans and both are commonly isolated from patients with caries. Since the role of C. albicans in caries remains unknown, our aim was to unravel this using an in vitro dual-species cariogenic oral biofilm model. Biofilms were grown for 24-72 h on glass cover slips or hydroxyapatite (HA) disks to mimic the surface of teeth. Medium pH, lactic acid production capacity and calcium release from HA disks were determined. All 24-h biofilms had external pH values below the critical pH of 5.5 where enamel dissolves. In contrast, 72-h dual-species biofilms had significantly higher pH (above the critical pH) and consequently decreased calcium release compared to single-species S. mutans biofilms. Counter intuitively, lactic acid production and growth of S. mutans were increased in 72-h dual-species biofilms. Candida albicans modulates the pH in dual-species biofilms to values above the critical pH where enamel dissolves. Our results suggest that C. albicans is not by definition a cariogenic microorganism; it could prevent caries by actively increasing pH preventing mineral loss.
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http://dx.doi.org/10.1093/femspd/ftw039DOI Listing
July 2016

Fine-Tuning Covalent Inhibition of Bacterial Quorum Sensing.

Chembiochem 2016 05 15;17(9):825-35. Epub 2016 Mar 15.

Department of Chemistry and, The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be'er Sheva, Israel.

Emerging antibiotic resistance among human pathogens has galvanized efforts to find alternative routes to combat bacterial virulence. One new approach entails interfering with the ability of bacteria to coordinate population-wide gene expression, or quorum sensing (QS), thus inhibiting the production of virulence factors and biofilm formation. We have recently developed such a strategy by targeting LasR, the master regulator of QS in the opportunistic human pathogen Pseudomonas aeruginosa, through the rational design of covalent inhibitors closely based on the core structure of the native ligand. We now report several groups of new inhibitors, one of which, fluoro-substituted ITC-12, displayed complete covalent modification of LasR, as well as effective QS inhibition in vitro and promising in vivo results. In addition to their potential clinical relevance, this series of synthetic QS modulators can be used as a tool to further unravel the complicated QS regulation in P. aeruginosa.
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http://dx.doi.org/10.1002/cbic.201500676DOI Listing
May 2016

Staphylococcus-Candida Interaction Models: Antibiotic Resistance Testing and Host Interactions.

Methods Mol Biol 2016 ;1356:153-61

Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Free University Amsterdam, Gustav Mahlerlaan 3004, 1081 LA, Amsterdam, The Netherlands.

The fungus Candida albicans and bacterium Staphylococcus aureus can coexist in polymicrobial biofilms. S. aureus attaches strongly to hyphae, but not to the yeast form, of C. albicans with important consequences for virulence. Hyphae-associated S. aureus is less susceptible to antibiotic treatment. Furthermore, co-inoculation of C. albicans and S. aureus causes more severe and widespread infection than either microorganism alone. In this chapter, a basic in vitro model for studying the interaction between C. albicans hyphae and S. aureus is presented, which makes use of a fluorescently labeled S. aureus strain. Furthermore, two protocols are described that allow investigation of the effect of C. albicans and S. aureus interaction on antibiotic susceptibility or on interactions with the host. The latter focuses on phagocytosis of C. albicans-adhered S. aureus by macrophages. The protocols presented here may serve as a starting point to study the interaction of C. albicans with various other bacterial species.
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http://dx.doi.org/10.1007/978-1-4939-3052-4_11DOI Listing
May 2016

In Vitro Models for Candida Biofilm Development.

Methods Mol Biol 2016 ;1356:95-105

Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Free University Amsterdam, Gustav Mahlerlaan 3004, 1081 LA, Amsterdam, The Netherlands.

Development of Candida spp. biofilms on medical devices such as catheters and voice prosthesis has been recognized as an increasing clinical problem. Different in vitro models are presented with increasing complexity. Each model system can be utilized for analysis of new active compounds to prevent or treat Candida biofilms as well as to study molecular processes involved in biofilm formation. Susceptibility studies of clinical isolates are generally performed in a simple 96-well model system similar to the CLSI standard. In the present chapter, optimized conditions that promote biofilm formation within individual wells of microtiter plates are described. In addition, the method has proven useful in preparing C. albicans biofilms for investigation by a variety of microscopic and molecular techniques. A more realistic and more complex biofilm system is presented by the Amsterdam Active Attachment (AAA) model. In this 24-well model all crucial steps of biofilm formation: adhesion, proliferation, and maturation, can be simulated on various surfaces, while still allowing a medium throughput approach. This model has been applied to study susceptibility, complex molecular mechanisms as well as interspecies (Candida-bacterium) interactions. Finally, a realistic microfluidics channel system is presented to follow dynamic processes in biofilm formation. In this Bioflux-based system, molecular mechanisms as well as dynamic processes can be studied at a high time-resolution.
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http://dx.doi.org/10.1007/978-1-4939-3052-4_8DOI Listing
May 2016

In vitro phenotypic differentiation towards commensal and pathogenic oral biofilms.

Biofouling 2015 ;31(6):503-10

a Top Institute Food and Nutrition , Wageningen , the Netherlands.

Commensal oral biofilms, defined by the absence of pathology-related phenotypes, are ubiquitously present. In contrast to pathological biofilms commensal biofilms are rarely studied. Here, the effect of the initial inoculum and subsequent growth conditions on in vitro oral biofilms was studied. Biofilms were inoculated with saliva and grown anaerobically for up to 21 days in McBain medium with or without fetal calf serum (FCS) or sucrose. Pathology-related phenotypes were quantified and the community composition was determined. Biofilms inoculated with pooled saliva or individual inocula were similar. Denaturing gradient gel electrophoresis (DGGE) analysis allowed differentiation of biofilms grown with sucrose, but not with FCS. Lactate production by biofilms was significantly increased by sucrose and protease activity by FCS. McBain grown biofilms showed low activity for both phenotypes. Three clinically relevant in vitro biofilm models were developed and could be differentiated based on pathology-related phenotypes but not DGGE analysis. These models allow analysis of health-to-disease shifts and the effectiveness of prevention measures.
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http://dx.doi.org/10.1080/08927014.2015.1067887DOI Listing
February 2016

Uses and limitations of green fluorescent protein as a viability marker in Enterococcus faecalis: An observational investigation.

J Microbiol Methods 2015 Aug 24;115:57-63. Epub 2015 May 24.

Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Department of Preventive Dentistry, Gustav Mahlerlaan 3004, 1081 LA Amsterdam, The Netherlands.

Enterococci are capable of producing biofilms that are notoriously difficult to treat and remove, for instance in root canal infections. The tenacious nature of these organisms makes screening of known and novel antimicrobial compounds necessary. While traditionally growth and fluorescence-based screening methods have proven useful, these methods have their limitations when applied to enterococci (e.g. time consuming, no kinetic data, diffusion properties of the fluorescent dyes). The aim of this study was to develop and validate a GFP-based high-throughput screening system to assess the bactericidal activity of a broad range of antimicrobial agents on Enterococcus faecalis and its biofilms. The effect of antimicrobial compounds on cell viability and GFP fluorescence of enterococcal planktonic and biofilm cells was determined using colony forming unit counts, fluorescence spectrophotometry and real-time imaging devices. There was a linear correlation between cell viability and GFP fluorescence. The intensity of the GFP signal was effected by the extracellular pH. For a range of antimicrobials however, there was no correlation between these two parameters. In contrast, for oxidizing agents such as sodium hypochlorite, the antimicrobial of choice for root canal disinfection, there was a correlation between loss of fluorescence and loss of viability. To conclude, the use of a GFP-based system to monitor the antimicrobial activity of compounds on E. faecalis is possible despite significant limitations. This approach is useful for analysis of susceptibility to oxidizing agents. Using real-time measuring devices to follow GFP fluorescence it should be possible to investigate the mode of action and rate of diffusion of oxidizing agents in E. faecalis biofilm.
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http://dx.doi.org/10.1016/j.mimet.2015.05.020DOI Listing
August 2015

LuxS signaling in Porphyromonas gingivalis-host interactions.

Anaerobe 2015 Oct 28;35(Pt A):3-9. Epub 2014 Nov 28.

Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam (ACTA), VU Free University and the University of Amsterdam, Gustav Mahlerlaan 3004, 1081 BT Amsterdam, The Netherlands. Electronic address:

Dental plaque is a multispecies biofilm in the oral cavity that significantly influences oral health. The presence of the oral anaerobic pathogen Porphyromonas gingivalis is an important determinant in the development of periodontitis. Direct and indirect interactions between P. gingivalis and the host play a major role in disease development. Transcriptome analysis recently revealed that P. gingivalis gene-expression is regulated by LuxS in both an AI-2-dependent and an AI-2 independent manner. However, little is known about the role of LuxS-signaling in P. gingivalis-host interactions. Here, we investigated the effect of a luxS mutation on the ability of P. gingivalis to induce an inflammatory response in human oral cells in vitro. Primary periodontal ligament (PDL) fibroblasts were challenged with P. gingivalis ΔluxS or the wild-type parental strain and gene-expression of pro-inflammatory mediators IL-1β, IL-6 and MCP-1 was determined by real-time PCR. The ability of P. gingivalis ΔluxS to induce an inflammatory response was severely impaired in PDL-fibroblasts. This phenotype could be restored by providing of LuxS in trans, but not by addition of the AI-2 precursor DPD. A similar phenomenon was observed in a previous transcriptome study showing that expression of PGN_0482 was reduced in the luxS mutant independently of AI-2. We therefore also analyzed the effect of a mutation in PGN_0482, which encodes an immuno-reactive, putative outer-membrane protein. Similar to P. gingivalis ΔluxS, the P. gingivalis Δ0482 mutant had an impaired ability to induce an inflammatory response in PDL fibroblasts. LuxS thus appears to influence the pro-inflammatory responses of host cells to P. gingivalis, likely through regulation of PGN_0482.
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http://dx.doi.org/10.1016/j.anaerobe.2014.11.011DOI Listing
October 2015

Systemic Staphylococcus aureus infection mediated by Candida albicans hyphal invasion of mucosal tissue.

Microbiology (Reading) 2015 Jan 20;161(Pt 1):168-181. Epub 2014 Oct 20.

Department of Microbiology and Immunology, School of Medicine, University of Maryland - Baltimore, 660 W. Redwood Street, Baltimore, MD 21201, USA.

Candida albicans and Staphylococcus aureus are often co-isolated in cases of biofilm-associated infections. C. albicans can cause systemic disease through morphological switch from the rounded yeast to the invasive hyphal form. Alternatively, systemic S. aureus infections arise from seeding through breaks in host epithelial layers although many patients have no documented portal of entry. We describe a novel strategy by which S. aureus is able to invade host tissue and disseminate via adherence to the invasive hyphal elements of Candida albicans. In vitro and ex vivo findings demonstrate a specific binding of the staphylococci to the candida hyphal elements. The C. albicans cell wall adhesin Als3p binds to multiple staphylococcal adhesins. Furthermore, Als3p is required for C. albicans to transport S. aureus into the tissue and cause a disseminated infection in an oral co-colonization model. These findings suggest that C. albicans can facilitate the invasion of S. aureus across mucosal barriers, leading to systemic infection in co-colonized patients.
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http://dx.doi.org/10.1099/mic.0.083485-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4274785PMC
January 2015

AI-2 of Aggregatibacter actinomycetemcomitans inhibits Candida albicans biofilm formation.

Front Cell Infect Microbiol 2014 21;4:94. Epub 2014 Jul 21.

Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Free University Amsterdam Amsterdam, Netherlands.

Aggregatibacter actinomycetemcomitans, a Gram-negative bacterium, and Candida albicans, a polymorphic fungus, are both commensals of the oral cavity but both are opportunistic pathogens that can cause oral diseases. A. actinomycetemcomitans produces a quorum-sensing molecule called autoinducer-2 (AI-2), synthesized by LuxS, that plays an important role in expression of virulence factors, in intra- but also in interspecies communication. The aim of this study was to investigate the role of AI-2 based signaling in the interactions between C. albicans and A. actinomycetemcomitans. A. actinomycetemcomitans adhered to C. albicans and inhibited biofilm formation by means of a molecule that was secreted during growth. C. albicans biofilm formation increased significantly when co-cultured with A. actinomycetemcomitans luxS, lacking AI-2 production. Addition of wild-type-derived spent medium or synthetic AI-2 to spent medium of the luxS strain, restored inhibition of C. albicans biofilm formation to wild-type levels. Addition of synthetic AI-2 significantly inhibited hypha formation of C. albicans possibly explaining the inhibition of biofilm formation. AI-2 of A. actinomycetemcomitans is synthesized by LuxS, accumulates during growth and inhibits C. albicans hypha- and biofilm formation. Identifying the molecular mechanisms underlying the interaction between bacteria and fungi may provide important insight into the balance within complex oral microbial communities.
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http://dx.doi.org/10.3389/fcimb.2014.00094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4104835PMC
March 2015
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