Lijian Jin

Biofilm lifestyle of Candida: a mini review

Candida is the major fungal pathogen of humans causing a variety of afflictions ranging from superficial mucosal diseases to deep seated mycoses. Biofilm formation is a major virulence factor in the pathogenicity of Candida, and Candida biofilms are difficult to eradicate especially because of their very high antifungal resistance. Consequently, research into the pathogenicity of Candida has focused on the prevention and management of biofilm development, their architecture, and antifungal resistance. Although studies have shed some light, molecular mechanisms that govern biofilm formation and pathogenicity still await full clarification. This review outlines the key features of what is currently known of Candida biofilm development, regulation and antifungal resistance and, their proteomics.

Oral mucosal fungal infections

This review has provided an overview of variants of oral mucosal candidiasis and current therapeutic techniques followed by an outline of the rare oral mycoses and their management. The advent of the HIV infection and the increasing prevalence of compromised individuals in the community as a consequence of surgical and medical advances have resulted in a resurgence of opportunistic infections, including oral candidiasis and other rare mycoses that were once considered exotic. It is now recognized that oral candidiasis may present in many clinical guises, including the classic white lesion of thrush as well in as nondescript, red mucosal lesions that may confound the unwary clinician. Other mycotic diseases, such as aspergillosis, cryptococcosis, histoplasmosis and mucormycosis, may manifest intra-orally, both as primary lesions and as secondary manifestations of systemic disease. Periodontal manifestations of mycotic diseases are rare. If at all, such lesions may present as erythematous areas as in linear gingival erythema and rarely as ulcerations in exotic mycoses. Most of the oral mycoses respond well to either topical or systemic therapy with the polyenes or azoles. In general, the management of oral fungal infections has been revolutionized by the triazole group of drugs, fluconazole and itraconazole, although recent reports indicate an alarming increase of resistant organisms, in particular to fluconazole.

Microbiology of Odontogenic Bacteremia: beyond Endocarditis

SUMMARY The human gingival niche is a unique microbial habitat. In this habitat, biofilm organisms exist in harmony, attached to either enamel or cemental surfaces of the tooth as well as to the crevicular epithelium, subjacent to a rich vascular plexus underneath. Due to this extraordinary anatomical juxtaposition, plaque biofilm bacteria have a ready portal of ingress into the systemic circulation in both health and disease. Yet the frequency, magnitude, and etiology of bacteremias due to oral origin and the consequent end organ infections are not clear and have not recently been evaluated. In this comprehensive review, we address the available literature on triggering events, incidence, and diversity of odontogenic bacteremias. The nature of the infective agents and end organ infections (other than endocarditis) is also described, with an emphasis on the challenge of establishing the link between odontogenic infections and related systemic, focal infections.

Architectural analysis, viability assessment and growth kinetics of Candida albicans and Candida glabrata biofilms

The human fungal pathogen Candida is able to form biofilms in almost all the medical devices in current use. Indeed, biofilm formation is a major virulence attribute of microorganisms and account for a majority of human infections. Therefore, understanding processes appertaining to biofilm development is an important prerequisite for devising new strategies to prevent or eradicate biofilm-related infections. In the present study we used an array of both conventional and novel analytical tools to obtain a comprehensive view of Candida biofilm development. Enumeration of colony forming units, colorimetric (XTT) assay, Scanning Electron Microscopy (SEM) and novel Confocal Laser Scanning Microscopy (CLSM) coupled with COMSTAT software analyses were utilised to evaluate growth kinetics; architecture and viability of biofilms of a reference (ATCC) and a clinical strain each of two Candida species, C. albicans and C. glabrata. Biofilm growth kinetics on a polystyrene substrate was evaluated from the initial adhesion step (1.5 h) up to 72 h. These analyses revealed substantial inter- and intra-species differences in temporal organisation of Candida biofilm architecture, spatiality and cellular viability, while reaching maturity within a period of 48 h, on a polystyrene substrate. There were substantial differences in the growth kinetics upon methodology, although general trend seemed to be the same. Detailed architectural analysis provided by COMSTAT software corroborated the SEM and CSLM views. These analyses may provide a strong foundation for down stream molecular work of fungal biofilms.

Cell Density and Cell Aging as Factors Modulating Antifungal Resistance of Candida albicans Biofilms

ABSTRACT Biofilm formation is a major virulence attribute of Candida pathogenicity which contributes to higher antifungal resistance. We investigated the roles of cell density and cellular aging on the relative antifungal susceptibility of planktonic, biofilm, and biofilm-derived planktonic modes of Candida. A reference and a wild-type strain of Candida albicans were used to evaluate the MICs of caspofungin (CAS), amphotericin B (AMB), nystatin (NYT), ketoconazole (KTC), and flucytosine (5FC). Standard, NCCLS, and European Committee on Antibiotic Susceptibility Testing methods were used for planktonic MIC determination. Candida biofilms were then developed on polystyrene wells, and MICs were determined with a standard 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide assay. Subsequently, antifungal susceptibility testing was performed for greater inoculum concentrations and 24- and 48-h-old cultures of planktonic Candida. Furthermore, Candida biofilm-derived planktonic cells (BDPC) were also subjected to antifungal susceptibility testing. The MICs for both C. albicans strains in the planktonic mode were low, although on increasing the inoculum concentration (up to 1 × 108 cells/ml), a variable MIC was noted. On the contrary, for Candida biofilms, the MICs of antifungals were 15- to >1,000-fold higher. Interestingly, the MICs for BDPC were lower and were similar to those for planktonic-mode cells, particularly those of CAS and AMB. Our data indicate that higher antifungal resistance of Candida biofilms is an intrinsic feature possibly related to the biofilm architecture rather than cellular density or cellular aging.

Coculture of Dental Pulp Stem Cells with Endothelial Cells Enhances Osteo-/Odontogenic and Angiogenic Potential In Vitro

INTRODUCTION Dental pulp stem cells (DPSCs) have received much attention as a promising population of stem cells in regenerative endodontics. Securing a good blood supply during regeneration is a challenging task because of the constricted apical canal opening, which allows only a limited blood supply. The aim of this study was to investigate any potential synergistic effects of dental pulp stem cells and endothelial cells (ECs) on osteo-/odontogenic and angiogenic differentiation in vitro. METHODS Different ratios of DPSCs and ECs were cultured in direct contact using optimized medium for coculture. The 70% confluent cocultures were incubated in the osteo-/odontogenic differentiation medium for up to 3 weeks. Alkaline phosphatase (ALP) activity, the expression levels of ALP, bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP) genes, and alizarin red staining for mineralization at different time points were analyzed. The tubular network formation on Matrigel and the gene expression levels of CD117, VEGF, CD34, and Flk-1 were used as assays to analyze angiogenesis. RESULTS The quantification of ALP in DPSC:EC cocultures revealed a greater ALP activity compared with DPSC-alone cultures. At all the time points, 1:1 cultures showed a significantly greater ALP activity than that of DPSC-alone cultures. Alizarin red staining and quantification revealed a much greater amount of calcification in the 1:1 and 1:5 cocultures compared with other cultures (P < .01). The expression levels of ALP, BSP, and DSPP genes further confirmed the greater osteo-/odontogenic differentiation in cocultures compared with those of DPSC-alone cultures. Matrigel assay showed that the addition of DPSCs stabilized preexisting vessel-like structures formed by ECs and increased the longevity of them. CONCLUSIONS Direct coculture of DPSCs and ECs enhances the in vitro differentiation toward osteo-/odontogenic and angiogenic phenotypes.

Exploring the oral bacterial flora: current status and future directions

OBJECTIVE The oral cavity forms an indispensable part of the human microbiome, for its unique and diverse microflora distributed within various niches. While majority of these organisms exhibit commensalism, shifts in bacterial community dynamics cause pathological changes within oral cavity and distant sites. The aim of this review was to appraise the current and emerging methods of detecting bacteria of the oral cavity paying particular attention to the cultivation independent methods. DESIGN Literature pertaining to cultivation based and cultivation independent methods of oral bacterial identification was reviewed. METHODS The specific advantages and disadvantages of cultivation based, microscopic, immunological and metagenomic identification methods were appraised. RESULTS Because of their fastidious and exacting growth requirements, cultivation based studies grossly underestimate the extent of bacterial diversity in these polymicrobial infections. Culture independent methods deemed more sensitive in identifying difficult to culture and novel bacterial species. CONCLUSION Apart from characterizing potentially novel bacterial species, the nucleic acid sequence data analyzed using various bioinformatics protocols have revealed that there are in excess of 700 bacterial species inhabiting the mouth. Moreover, the latest pyrosequencing based methods have further broadened the extent of bacterial diversity in oral niches.

Candida albicans biofilm formation is associated with increased anti‐oxidative capacities

Candida albicans is a common, opportunistic, human fungal pathogen that causes a variety of mucosal and systemic afflictions. It exists in nature both in the biofilm or the sessile phase, as well as in the free‐floating or the planktonic phase. Candida biofilms, in particular, display unique characteristics that confer survival advantages over their planktonic counterparts, such as their recalcitrance to common antifungals. The mechanisms underlying Candida biofilm formation and their attributes are poorly understood. In this study, we used a 2‐DE‐based approach to characterize the protein markers that are differentially expressed in Candida biofilms in comparison to their planktonic counterparts. Using tandem mass spectrometric analysis, we have identified a significant number of proteins including alkyl hydroperoxide reductase, thioredoxin peroxidase, and thioredoxin involved in oxidative stress defenses that are upregulated in the biofilm phase. These proteomic findings were further confirmed by real‐time PCR and lucigenin‐based chemiluminescence assays. In addition, we demonstrate that a drug target for the new antifungal agent echinocandin, is abundantly expressed and significantly upregulated in Candida biofilms. Taken together, these data imply that the biofilm mode, Candida, compared with their planktonic counterparts, exhibits traits that can sustain oxidative stress (anti‐oxidants), and thereby exert resistance to commonly used antifungals.

Pseudomonas aeruginosa inhibits in-vitro Candida biofilm development

BackgroundElucidation of the communal behavior of microbes in mixed species biofilms may have a major impact on understanding infectious diseases and for the therapeutics. Although, the structure and the properties of monospecies biofilms and their role in disease have been extensively studied during the last decade, the interactions within mixed biofilms consisting of bacteria and fungi such as Candida spp. have not been illustrated in depth. Hence, the aim of this study was to evaluate the interspecies interactions of Pseudomonas aeruginosa and six different species of Candida comprising C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, and C. dubliniensis in dual species biofilm development.ResultsA significant reduction in colony forming units (CFU) of C. parapsilosis (90 min), C. albicans and C. tropicalis (90 min, 24 h and 48 h), C. dubliniensis and C. glabrata, (24 h and 48 h) was noted when co-cultured with P. aeruginosa in comparison to their monospecies counterparts (P < 0.05). A simultaneous significant reduction in P. aeruginosa numbers grown with C. albicans (90 min and 48 h), C. krusei (90 min, 24 h and 48 h),C. glabrata, (24 h and 48 h), and an elevation of P. aeruginosa numbers co-cultured with C. tropicalis (48 h) was noted (P < 0.05). When data from all Candida spp. and P. aeruginosa were pooled, highly significant mutual inhibition of biofilm formation was noted (Candida P < 0.001, P. aeruginosa P < 0.01). Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM) analyses confirmed scanty architecture in dual species biofilm in spite of dense colonization in monospecies counterparts.ConclusionsP. aeruginosa and Candida in a dual species environment mutually suppress biofilm development, both quantitatively and qualitatively. These findings provide a foundation to clarify the molecular basis of bacterial-fungal interactions, and to understand the pathobiology of mixed bacterial-fungal infections.

Porphyromonas gingivalis lipopolysaccharide lipid A heterogeneity differentially modulates the expression of IL‐6 and IL‐8 in human gingival fibroblasts

AIM Porphyromonas gingivalis lipopolysaccharide (LPS) displays a significant amount of structural heterogeneity, containing both tetra- (LPS(1435/1449) ) and penta-acylated (LPS(1690) ) lipid A structures. This study investigated the effects of the two isoforms of P. gingivalis LPS on the expression of IL-6, IL-8 and TNF-α in human gingival fibroblasts (HGFs). MATERIALS AND METHODS HGFs were stimulated with P. gingivalis LPS(1435/1449) and LPS(1690) in both dose- (1 ng-10 μg/ml) and time-dependent (2-48 h) experiments. Total RNA and protein were extracted and used for analysis of the IL-6, IL-8 and TNF-α transcripts as well as IL-6 and IL-8 proteins, by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS P. gingivalis LPS(1690) significantly up-regulated the mRNA and protein expression of IL-6 and IL-8, whereas P. gingivalis LPS(1435/1449) did not induce significant host response. The expression levels of IL-6 and IL-8 up-regulated by P. gingivalis LPS(1690) continuously increased with time course. In contrast, TNF-α transcript expression was up-regulated promptly by P. gingivalis LPS(1690) after 2 h of stimulation and gradually declined afterwards. CONCLUSIONS This study suggests that P. gingivalis LPS heterogeneity may differentially modulate the pro-inflammatory cytokine expression in HGFs, which may contribute to periodontal pathogenesis.