Isolation, identification and characterization of L actobacillus species diversity from Meekiri: traditional fermented buffalo milk gels in Sri Lanka.

Traditional fermented buffalo milk gel; Meekiri, is a popular buffalo milk-derived product in Sri Lanka. Predominantly, it is produced using the back-slopping (adding a small amount of the previous fermentate) technique, following the life-long traditions available at the cottage level. Hence, diverse and unclassified starter cultures are likely to be established across the varying geographical regions of Meekiri production. In the present study, we aimed to elucidate the diversity of lactic acid bacteria (LAB) and their characteristics including probiotic properties from major Meekeri production areas (n = 22) in Sri Lanka. Lactic acid bacteria was isolated from locally produced Meekiri samples (n = 23) and characterized based on morphological, biochemical, physiological profiles and potential of probiotic properties. The isolates revealed five different colony and cell morphologies and were classified as heterofermenters, homofermenters and facultative heterofermenters based on CO2 production using glucose. None of the isolates showed the ability to grow either at 5 °C or 0 °C, while 71 % and 100 % survival of the isolates were observed at 15 °C and 45 °C, respectively. Amplified ribosomal DNA restriction analysis (ARDRA) primarily grouped the isolates into three distinct clusters based on their DNA banding patterns. Subsequently, 16S rRNA sequencing of isolates revealed the presence of four species namely, Limosilactobacillus fermentum (n = 18), Latilactobacillus curvatus (n = 2), Lactobacillus acidophilus (n = 2) and Lactiplantibacillus plantarum (n = 1) and in the phylogenetic analysis, it was represented by four distinctive clades. All the isolated species demonstrated promising probiotic potential with antibiotic sensitivity, antimicrobial properties, bile acid tolerance and acid tolerance. In conclusion, traditional back-slopping Meekiri in Sri Lanka contains diverse LAB, with a negligible geographical variation at species-level. Our work provides a strong foundation and insights into future applications in starter culture development for the fermentation of buffalo's milk.

Candida-Bacteria Interactions: Their Impact on Human Disease.

Candida species are the most common infectious fungal species in humans; out of the approximately 150 known species, Candida albicans is the leading pathogenic species, largely affecting immunocompromised individuals. Apart from its role as the primary etiology for various types of candidiasis, C. albicans is known to contribute to polymicrobial infections. Polymicrobial interactions, particularly between C. albicans and bacterial species, have gained recent interest in which polymicrobial biofilm virulence mechanisms have been studied including adhesion, invasion, quorum sensing, and development of antimicrobial resistance. These trans-kingdom interactions, either synergistic or antagonistic, may help modulate the virulence and pathogenicity of both Candida and bacteria while uniquely impacting the pathogen-host immune response. As antibiotic and antifungal resistance increases, there is a great need to explore the intermicrobial cross-talk with a focus on the treatment of Candida-associated polymicrobial infections. This article explores the current literature on the interactions between Candida and clinically important bacteria and evaluates these interactions in the context of pathogenesis, diagnosis, and disease management.

A review of the ultrastructural features of superficial candidiasis.

Commensal yeast Candida causes opportunistic infections ranging from superficial lesions to disseminated mycoses in compromised patients. Superficial candidiasis, the commonest form of candidal infections, primarily affects the mucosa and the skin where Candida lives as a commensal. Conversion of candidal commensalism into opportunism at the fungal-epithelial interface is still ill-defined. Nevertheless, fungal virulence mechanisms such as adhesion to epithelia, morphogenesis, production of secretory hydrolytic enzymes, and phenotypic switching are thought to contribute in the process of pathogenesis. On the other hand, host responses in terms of immunity and local epithelial responses are actively involved in resisting the fungal challenge at the advancing front of the infection. Ultrastructural investigations using electron microscopy along with immunohistochemistry, cytochemistry, etc. have helped better viewing of Candida-host interactions. Thus, studies on the ultrastructure of superficial candidiasis have revealed a number of fungal behaviors and associated host responses such as adhesion, morphogenesis (hyphae and appresoria formation), thigmotropism, production and distribution of extracellular enzymes, phagocytosis, and epithelial changes. The purpose of this review is to sum up most of the ultrastructural findings of Candida-host interactions and to delineate the important pathological processes underlying superficial candidiasis.

Experimental superficial candidiasis on tissue models.

Candida species are common pathogens causing superficial mycoses primarily affecting the mucosa and the skin in humans. Crucial steps during pathogenesis of superficial candidiasis comprise fungal adhesion, colonisation and subsequent penetration of the respective tissues. Exploring these pathological events and perhaps fungal and tissue responses towards drug treatment is imperative in the management of this infection. Unfortunately, pathological biopsies of superficial candidiasis do not exhibit the early changes in the host-pathogen interaction as the tissues are already invaded by the fungi. In vivo experimental assessments of pathological processes of superficial candidiasis are also limited because of the difficulties in providing reproducible and comparable conditions in the host environment. Conversely, in vitro models have helped studying fungal-host interactions under more defined and controlled conditions. Some common in vitro models used to simulate superficial candidiasis are chick chorioallantoic membrane, mucosal explants and single layer or multiple layer cell cultures. Interestingly, these experimental approaches share advantages as well as disadvantages when compared with in vivo conditions. Hence, this review intends to discuss about the experimental superficial candidiasis produced in various tissue models and their advantages as well as disadvantages with a particular reference to further improvement of validity and reliability of such experiments.

The post-antifungal effect (PAFE) of amphotericin B, nystatin, ketoconazole and 5-fluorocytosine and its impact on the colonization traits of Candida glabrata.

The post-antifungal effect (PAFE) has been shown to affect Candida pathogenicity, but there is little information on either PAFE or its association with the colonization traits of Candida glabrata. The objective of this study was to determine, in vitro, the PAFE on 14 C. glabrata isolates following exposure to amphotericin B (AMB), nystatin (NYS), ketoconazole (KETO) and 5-fluorocytosine (5FC). In addition, we evaluated the impact of PAFE on yeast adherence to buccal epithelial cells (BEC), cell-surface-hydrophobicity (CSH) and biofilm growth (BG) on denture acrylic surfaces. PAFE was induced following a 1-h exposure of yeasts to (x1-x4MIC) of AMB, NYS, KETO and 5FC in RPMI medium and, measured using automated turbidometry. The BEC adhesion, CSH and BG assays were performed by the methods of Kimura & Pearsall, Sweet et al., and Jin et al., respectively. Significant differences in PAFE (P < 0.001) were observed after exposure to AMB and NYS, but not KETO and 5FC. Following exposure to AMB, NYS, KETO and 5FC, significant inter-strain differences (P < 0.001) were observed in percentage terms in adhesion (39.0%, 43.48%, 38.28%, 35.07%) and biofilm growth (42.86%, 39.86%, 42.81%, 36.38%), respectively. Short exposure of C. glabrata to sub-cidal concentrations of antifungals modulates yeast growth and also affects some of their colonization traits.

Candidal onychomycosis: a mini-review.

Onychomycosis is a common fungal infection affecting nails. The primary cause for onychomycosis is dermatophytes, while Candida species have emerged as second-line pathogens. Onychomycosis due to Candida (candidal onychomycosis) is increasingly found in individuals having defective immunity consequential to aging, diabetes mellitus, vascular diseases, HIV infection and drug therapies such as immunosuppressives and broad-spectrum antibiotics. Breached local immunity at the nail complex due to trauma, chronic exposure to moisture and chemicals including smoke, detergents, soap, etc., also contribute to candidal onychomycosis. Adhesion, filamentation, secretion of extracellular enzymes and the development of antifungal resistance are some of the virulence mechanisms of Candida species associated with onychomycosis. Diagnosis of onychomycosis depends on history and clinical examination, direct microscopic investigation, mycological culture and histopathology. Restoration of immune defenses, elimination of fungi using appropriate drug therapy and improvement of nail hygiene with the removal of predisposing factors are key aspects in the management of candidal onychomycosis.

Effect of filamentation and mode of growth on antifungal susceptibility of Candida albicans.

Biofilm formation involving profuse hyphal growth is a major characteristic of Candida spp. and confers higher antifungal resistance than its planktonic mode of growth. We investigated the antifungal susceptibility of Candida albicans and its hyphal mutants (Delta efg1/efg1, Delta cph1/cph1 and DeltaDelta cph1/cph1 efg1/efg1) to commonly used antifungals during planktonic, adhesion and biofilm modes of growth. The minimum inhibitory concentration (MIC) of each antifungal agent was determined for a lower inoculum (1x10(3) cells/mL) and higher inoculum (1x10(7) cells/mL) of planktonic Candida. Furthermore, MICs of C. albicans biofilms and adhesion modes of growth were determined with a standard XTT assay. Candida albicans in adhesion and biofilm modes of growth, but not in planktonic mode, were resistant to all five antifungal agents tested. Although Delta efg1/efg1 and DeltaDelta cph1/cph1 efg1/efg1 mutants formed less biofilm than wild-type C. albicans SC5314, they were similarly resistant to caspofungin. However, these mutants were more sensitive to amphotericin B and nystatin than the wild-type. Adhesion per se confers increased resistance to antifungal agents, which is further pronounced in the biofilm mode of Candida. Filamentation does not appear to be a major determinant of the antifungal resistance in Candida biofilms.

A comparative study of candidal invasion in rabbit tongue mucosal explants and reconstituted human oral epithelium.

The purpose of this study is to compare the light and scanning electron microscopic (SEM) features of tissue invasion by three Candida species (C. albicans, C. tropicalis, and C. dubliniensis) in two different tissue culture models: rabbit tongue mucosal explants (RTME) and reconstituted human oral epithelium (RHOE). Tongue mucosal biopsies of healthy New Zealand rabbits were maintained in explant culture using a transwell system. RHOE was obtained from Skinethic Laboratory (Nice, France). RTME and RHOE were inoculated with C. albicans, C. tropicalis, and C. dubliniensis separately and incubated at 37 degrees C, 5% CO(2), and 100% humidity up to 48 h. Light microscopic and SEM examinations of uninfected (controls) and infected tissues were performed at 24 and 48 h. C. albicans produced characteristic hallmarks of pathological tissue invasion in both tissue models over a period of 48 h. Hyphae penetrated through epithelial cells and intercellular gaps latter resembling thigmotropism. SEM showed cavitations on the epithelial cell surfaces particularly pronounced at sites of hyphal invasion. Some hyphae on RTME showed several clusters of blastospores attached in regular arrangements resembling "appareil sporifere". C. tropicalis and C. dubliniensis produced few hyphae mainly on RTME but they did not penetrate either model. Our findings indicate that multiple host-fungal interactions such as cavitations, thigmotropism, and morphogenesis take place during candidal tissue invasion. RTME described here appears to be useful in investigations of such pathogenic processes of Candida active at the epithelial front.

Prevalence of oral Candida species in leprosy patients from Cambodia and Thailand.

BACKGROUND: Leprosy is a chronic bacterial infection which may lead to significant orofacial morbidity. However, reports on the oral mycotic flora of leprosy patients are rare. The aim of the current study was to explore the oral yeast carriage in two groups of leprosy patients. METHODS: 40 Cambodian (seven men, 33 women) and 48 Thai (14 men, 34 women) leprosy patients from Leprosy Rehabilitation Centre Khien Kleang, Phnom Penh, Cambodia and McKean Rehabilitation Center, Chiangmai, Thailand were randomly selected and their demographic data and clinical history were recorded. Tongue and palatal swabs of each patient were collected using sterile Fungi-Quick swabs (Hain Diagnostika, Nehren, Germany) and they were cultured aerobically on Sabouraud's dextrose agar and CHROMAgar (CHROMagar, Paris, France). Yeast were identified by germ tube, chlamydospore production, and assimilation tests (API 20C AUX, Bio-Merieux, Marcy l'Etoile, France) and reconfirmed using APILAB Plus system (Bio-Merieux). RESULTS: Two groups (Cambodian and Thai) had median age of 35 and 64 years. They had been with leprosy for median durations of 17.7 and 38.9 years (P<0.05), respectively. Overall yeast carriage in two cohorts were 80% and 93.75%. Candida albicans had highest carriage rate in either group (65.6%, 44.4%). Candida krusei and C. glabrata existed as second-line colonizers after C. albicans. Candida glabrata carriage was significantly higher in Thai patients (P<0.05). Multispecies carriage was seen in three Cambodian (9.4%) and five Thai (11.5%) patients. CONCLUSIONS: This study indicates high oral yeast carriage in leprosy patients. Candida albicans remains predominant while C. krusei and C. glabrata are second-line oral colonizers. Co-inhabitation of multiple yeast species is also noted in these patients' oral mycotic flora.

IL-1alpha, IL-1ra and IL-8 are differentially induced by Candida in experimental oral candidiasis.

OBJECTIVE: To investigate the expression of interleukin-1alpha (IL-1alpha), IL-1ra and IL-8 by the oral epithelium challenged by various Candida species. MATERIALS AND METHODS: In vitro candidiasis was induced by C. albicans wild type SC5314, its EFG1, CPH1 and secretory aspartyl proteinase (SAP) mutants and, ATCC isolates of C. albicans, C. tropicalis and C. dubliniensis using a reconstituted human oral epithelium (RHOE) model. IL-1alpha, IL-1ra and IL-8 levels in culture media were quantified by an enzyme-linked immunosorbent assay at 12, 24 and 48 h. Fungal invasion and IL-1ra expression in RHOE were detected by periodic acid-Schiff staining and immunohistochemistry. RESULTS: Overall, the invasive Candida induced relatively higher levels of IL-1alpha, IL-1ra and IL-8 in the culture media than the noninvasive isolates. IL-1alpha and IL-1ra levels induced by Candida with hyphal invasion were significantly higher (P < 0.05) than those induced by the isolates without hyphal invasion at 12, 24 and 48 h. Candida albicans SC5314 induced IL-1ra expression in RHOE at 12 and 24 h but not at 48 h consistent with its hyphal invasion; while the noninvasive mutants and non-albicans Candida induced IL-1ra expression at 48 h. CONCLUSIONS: The cytokine expression profiles in experimental oral candidiasis may be associated with the invasive potential of Candida.

Quantitative evaluation of tissue invasion by wild type, hyphal and SAP mutants of Candida albicans, and non-albicans Candida species in reconstituted human oral epithelium.

BACKGROUND: Oral candidiasis is a common problem in compromised patients. Although several non-albicans Candida species have emerged as pathogens the majority of candidal infections are caused by Candida albicans. Morphogenesis from the blastospore to filamentous phase, and production of secretory aspartyl proteinases (SAP) are two major virulence attributes of these opportunistic yeast. Histopathology of oral candidiasis is characterized by fungal invasion of the superficial epithelium although the invasive potentials of different Candida species vary. Computerized image analysis systems (IAS) utilizing immunohistochemistry have been successfully employed for quantification of such histopathological features. The purpose of this study was to evaluate quantitatively the in vitro invasive potential of C. albicans and its hyphal and SAP mutants, and five other non-albicans Candida species using a computerized IAS. METHODS: In vitro human oral candidiasis was produced using five wild type and one reference C. albicans isolates, hyphal and SAP mutants of C. albicans SC 5314, and one wild type and one reference isolate each of C. tropicalis, C. dubliniensis, C. glabrata, C. parapsilosis and C. krusei in a reconstituted human oral epithelium (RHOE) model. The infected tissues were examined histologically at 12, 24 and 48 h. Invading fungal elements were visualized by periodic acid-Schiff (PAS) staining and quantitatively evaluated as a percentage of total tissue invasive area, using a computerized IAS. RESULTS: All C. albicans isolates including hyphal mutant cph1/cph1 and SAP mutants; sap 1-3, sap 4-6 produced hyphae and differentially (P < 0.05) invaded the tissue over 48 h. The invasive potential of hyphal mutant cph1/cph1 and SAP mutants (sap 1-3, sap 4-6) were similar to the parent wild-type isolate at 12 h although after 24 h their invasion was dissimilar (P < 0.05). Non-albicans Candida species and hyphal mutants; efg1/efg1, efg1/efg1 cph1/cph1 were all non-invasive. CONCLUSIONS: RHOE model in combination with computerized image analysis permits for the first time, the assessment of invasive potential of Candida species in a quantitative manner. The differential tissue invasive patterns of various C. albicans isolates, their mutants and other Candida species are also described.

Differential phospholipase gene expression by Candida albicans in artificial media and cultured human oral epithelium.

Phospholipases B1, B2, C and D of Candida albicans play a significant role in the host invasive process. Hence we evaluated the in vitro expression of PLB1, PLB2, PLC1 and PLD1 in phospholipase-positive (PL(+)) and -deficient (PL(-)) C. albicans isolates in egg yolk agar (EYA), yeast peptone dextrose broth (YPD), and in a model of oral candidiasis based on reconstituted human oral epithelium (RHOE). The growth of Candida was then determined in YPD and its cellular invasion was investigated using the RHOE model. The PL(+) group demonstrated PLB1, PLB2, PLC1 and PLD1 expression in both EYA and YPD, in contrast to the PL(-) group, which expressed only PLB2 and PLD1. Although PL(+) isolates grew profusely in the RHOE model, they expressed only PLB2, PLC1 and PLD1, and not PLB1. Gene expression investigations could not be carried out with PL(-) isolates due to their inability to grow in the RHOE model. Significant growth differences in YPD medium were also observed within the PL(+) and PL(-) groups. Taken together, these findings indicate that phospholipase gene expression in C. albicans is differentially affected by their growth milieu, and this in turn may modulate the disease outcomes in vivo.

An ultrastructural and a cytochemical study of candidal invasion of reconstituted human oral epithelium.

BACKGROUND: Opportunistic yeast, Candida albicans causes superficial and systemic mycoses in compromised patients. Adhesion to host tissues, morphogenesis and extracellular phospholipases (PL) are thought to contribute to its virulence. The nature of numerous host-parasite interactions at the invasive phase of oral candidiasis is not fully understood. Hence in this study, we explore the ultrastructural features of oral candidiasis using a tissue culture model based on reconstituted human oral epithelium (RHOE). METHODS: Reconstituted human oral epithelium (Skinethic Laboratory, Nice, France) was inoculated with C. albicans SC5314 and incubated up to 48 h. The infected tissue was harvested at 12, 24 and 48 h and examined using light, scanning (SEM) and transmission electron microscopy (TEM). Localized activity of PLs of C. albicans during tissue invasion was also examined using a cytochemical method. RESULTS: Over a period of 48 h C. albicans invaded the RHOE, and histological examination revealed characteristic hallmarks of pathological tissue invasion. Hyphal penetration into the superficial epithelium, particularly at cell junctions, together with features of cellular internalization of yeasts was noted. Phospholipase activity was visible at the tips of hyphae and initial sites of bud formation. Further, SEM studies revealed cavitations on the surface epithelial cells particularly pronounced at the sites of hyphal invasion. Hyphal invasion was seen both at cell surfaces and intercellular cell junctions of the epithelium, the latter resembling thigmotropic behaviour. CONCLUSIONS: Our findings confirm that multiple cellular interactions such as internalization, thigmotropism and extracellular PLs contribute to invasive candidiasis. The RHOE model, described here, appears to be a satisfactory model for the investigation of ultrastructural and histochemical features of invasive candidiasis in humans.