Development of a bis-pyrene phospholipid probe for fluorometric detection of phospholipase A2 inhibition.

In this work, we report the design, synthesis, and application of a bis-pyrene phospholipid probe for detection of phospholipase A2 action through changes in pyrene monomer and excimer fluorescence intensities. Continuous fluorometric assays enabled detection of the activities of multiple PLA2 enzymes as well as the decrease in catalysis by PLA2 from honey bee venom caused by the inhibitor p-bromo phenacylbromide. Thin-layer chromatography and mass spectrometry analysis were also used to validate probe hydrolysis by PLA2. Mass spectrometry data also supported cleavage of the probe by phospholipase C and D enzymes, although changes in fluorescence were not observed in these cases. Nevertheless, the bis-pyrene phospholipid probe developed in this work is effective for detection of PLA2 enzyme activity through an assay that enables screening for inhibitor development.

Secreted Hydrolytic and Haemolytic Activities of Malassezia Clinical Strains.

Malassezia yeasts are opportunistic pathogens associated with a number of skin diseases in animals and humans. The free fatty acids released through these organisms' lipase and phospholipase activities trigger inflammation in the host; thus, these lipase and phospholipase activities are widely recognised as some of the most important factors in Malassezia pathogenesis. In this study, we sought to investigate and examine the relationship between these secreted hydrolytic activities and haemolytic activity in newly isolated Malassezia clinical strains. This characterisation was expected to elucidate pathogenicity of this fungus. We isolated 35 clinical strains of Malassezia spp.; the most frequently isolated species were M. sympodialis and M. furfur. Next, we analysed the hydrolytic activities of all of these clinical isolates; all of these strains (except for one M. dermatis isolate) showed detectable lipase and phospholipase activities against 4-nitrophenyl palmitate and L-α-phosphatidylcholine, dipalmitoyl, respectively. Most of the M. globosa isolates showed higher lipase activities than isolates of other Malassezia species. In terms of phospholipase activity, no significant difference was observed among species of Malassezia, although one isolate of M. globosa showed considerably higher phospholipase activity than the others. All tested strains also exhibited haemolytic activity, both as determined using 5% (v/v) sheep blood agar (halo assay) and by quantitative assay. Although all tested strains showed detectable haemolytic activity, we did not observe an apparent correlation between the secreted lipase and phospholipase activities and haemolytic activity. We infer that the haemolytic activities of Malassezia spp. are mediated by non-enzymatic factor(s) that are present in the secreted samples.

Comparative analysis of proteinase, phospholipase, hydrophobicity and biofilm forming ability in Candida species isolated from clinical specimens.

Candida species are the commensal organisms of human and animal mucosa that cause a wide range of debilitating diseases in immunocompromised patients and other susceptible individuals. The present study aimed to investigate the ability of clinical isolates of various Candida species to produce proteinase and phospholipase, hydrophobicity and biofilm forming ability that assumed to have a vital role in Candida pathogenicity. Eighty-four Candida strains belonged to Candida albicans (44.1%), C. glabrata (5.9%), C. guilliermondii (5.9%), C. krusei (10.8%), C. parapsilosis (26.2%), and C. tropicalis (7.1%) were examined for proteinase and phospholipase production, cell surface hydrophobicity and biofilm forming ability. The production of proteinase and phospholipase was detected in 81 (96.4%) and 79 (94.1%) of the strains, respectively. C. albicans showed the highest proteinase and phospholipase activity (mean Pz values of 0.42±0.25 and 0.72±0.28) and biofilm formation ability (0.66±0.22). C. parapsilosis had the highest hydrophobicity (42.97±16.1), which showed a good correlation with biofilm formation ability. A considerable percentage of non-albicans Candida strains produced significant amounts of proteinase and phospholipase with a good ability of biofilm formation in vitro. Taken together, our results further substantiated that enzymatic activity, hydrophobicity and the ability for biofilm formation are important virulence factors which may be account for pathogenicity of various Candida species distributed in albicans and non-albicans groups.

Activities of Endogenous Lipase and Lipolysis Oxidation of Low-Salt Lactic Acid-Fermented Fish (Decapterus maruadsi).

There is increasing demand for low-salt meat products that retain traditional flavors. In this study, dry-salted fish (Decapterus maruadsi) were processed by 2 methods to obtain traditional salted fish (HS) and low-salt lactic acid-fermented fish (LAF). The relationship between lipolysis and lipid oxidation was investigated by evaluating changes in endogenous lipase (lipolytic enzymes; lipoxygenase, LOX), free fatty acid composition, thiobarbituric acid reactive substances (TBARS), and peroxide value (POV) during processing. Lipolytic enzyme activity showed a decreasing trend, in general. LOX activity initially increased and eventually decreased. Phospholipase, acid lipase, and neutral lipase activity was 0.33, 0.17, 0.57 (in HS) and 0.39, 0.25, 0.67 (in LAF) times in the final product than the activity levels observed in fresh fish. A principal component analysis indicated that phospholipase and neutral lipase play major roles in promoting lipid hydrolysis (in HS and LAF), the correlation between lipolytic activity and lipid oxidation in HS is greater than the correlation in LAF, and the contribution of LOX to lipid oxidation was minor in salted fish.

The frequency, antifungal susceptibility and enzymatic profiles of Candida species in cases of onychomycosis infection.

Although the frequency of candidal onychomycosis is increasing daily, there is little information in literature about the epidemiology, pathogenesis, and antifungal susceptibility of this dermatological disease. This study aimed to provide information about the epidemiology, pathogenesis, and azole susceptibility of Candida species isolated from patients living in a region with continental climate. After identification of the isolated strains using conventional methods, proteinase and phospholipase activities were determined by a plate method and biofilm-forming ability was determined using the microplate method. Susceptibility of the same species to fluconazole (FLU), voriconazole (VRC), miconazole (MNZ), itraconazole (ITZ), and ketoconazole (KTZ) were determined by microdilution method. The 50 Candida isolates included 23 C. parapsilosis (46%), 13 C. albicans (26%), 4 C. guilliermondii(8%), 4 C.tropicalis (8%), 2 C.krusei(2%), 1 C.lusitaniae (2%), 1 C. sake (2%), and 1 C. kefyr (2%) isolates. The geometric mean (GM) of the minimum inhibitory concentration (MIC) for FLU, KTZ, VRC, MNZ, and ITZ was 0.4 µg/mL, 0.08 µg/mL, 0.08 µg/mL, 0.2 µg/mL, and 0.6 µg/mL, respectively. Proteinase, phospholipase, and biofilm-forming ability were detected in 18%(9/50), 20%(10/50), and 6%(3/50) of the Candida isolates, respectively. We found that the most frequently isolated species is C.parapsilosis. On the basis of the GM values, the most effective azoles are ketoconazole and voriconazole. The isolated Candida species exhibited low phospholipase, proteinase, and biofilm formation activities.

Investigations of the prevalence and virulence of Candida albicans in periodontal and endodontic lesions in diabetic and normoglycemic patients.

OBJECTIVE: The aim of this study was to investigate the prevalence of isolated Candida albicans from periodontal endodontic lesions in diabetic and normoglycemic patients, and the fungi's virulence in different atmospheric conditions. MATERIAL AND METHODS: A case-control study was conducted on 15 patients with type 2 diabetes mellitus (G1) and 15 non-diabetics (G2) with periodontal endodontic lesions. Samples of root canals and periodontal pockets were plated on CHROMagar for later identification by polymerase chain reaction (PCR) and virulence test. RESULTS: C. albicans was identified in 79.2% and 20.8% of the 60 samples collected from diabetic and normoglycemic patients, respectively. Of the 30 samples collected from periodontal pockets, 13 showed a positive culture for C. albicans, with 77% belonging to G1 and 23% to G2. Of the 11 positive samples from root canals, 82% were from G1 and 18% from G2. Production of proteinase presented a precipitation zone Pz<0.63 of 100% in G1 and 72% in G2, in redox and negative (Pz=1), under anaerobic conditions in both groups. Hydrophobicity of the strains from G1 indicated 16.4% with low, 19.3% with moderate, and 64.3% with high hydrophobicity in redox. In G2, 42.2% had low, 39.8% had moderate, 18% had high hydrophobicity in redox. In anaerobic conditions, G1 showed 15.2% with low, 12.8% with moderate, and 72% with high hydrophobicity; in G2, 33.6% had low, 28.8% had moderate, and 37.6% had high hydrophobicity. There was statistical difference in the number of positive cultures between G1 and G2 (p<0.05) with predominance in G1. There was statistical difference for all virulence factors, except hemolysis (p=0.001). CONCLUSIONS: Candida albicans was isolated more frequently and had higher virulence in diabetic patients.

Investigations of the prevalence and virulence of Candida albicans in periodontal and endodontic lesions in diabetic and normoglycemic patients

Abstract Pulpal and periodontal tissues have similar microbiota that allows cross-contamination between the pulp and periodontal tissues. Objective The aim of this study was to investigate the prevalence of isolated Candida albicans from periodontal endodontic lesions in diabetic and normoglycemic patients, and the fungi's virulence in different atmospheric conditions. Material and Methods A case-control study was conducted on 15 patients with type 2 diabetes mellitus (G1) and 15 non-diabetics (G2) with periodontal endodontic lesions. Samples of root canals and periodontal pockets were plated on CHROMagar for later identification by polymerase chain reaction (PCR) and virulence test. Results C. albicans was identified in 79.2% and 20.8% of the 60 samples collected from diabetic and normoglycemic patients, respectively. Of the 30 samples collected from periodontal pockets, 13 showed a positive culture for C. albicans, with 77% belonging to G1 and 23% to G2. Of the 11 positive samples from root canals, 82% were from G1 and 18% from G2. Production of proteinase presented a precipitation zone Pz<0.63 of 100% in G1 and 72% in G2, in redox and negative (Pz=1), under anaerobic conditions in both groups. Hydrophobicity of the strains from G1 indicated 16.4% with low, 19.3% with moderate, and 64.3% with high hydrophobicity in redox. In G2, 42.2% had low, 39.8% had moderate, 18% had high hydrophobicity in redox. In anaerobic conditions, G1 showed 15.2% with low, 12.8% with moderate, and 72% with high hydrophobicity; in G2, 33.6% had low, 28.8% had moderate, and 37.6% had high hydrophobicity. There was statistical difference in the number of positive cultures between G1 and G2 (p<0.05) with predominance in G1. There was statistical difference for all virulence factors, except hemolysis (p=0.001). Conclusions Candida albicans was isolated more frequently and had higher virulence in diabetic patients.

Usefulness of the Non-conventional Caenorhabditis elegans Model to Assess Candida Virulence.

Invasive candidiasis is caused mainly by Candida albicans, but other Candida species have increasing etiologies. These species show different virulence and susceptibility levels to antifungal drugs. The aims of this study were to evaluate the usefulness of the non-conventional model Caenorhabditis elegans to assess the in vivo virulence of seven different Candida species and to compare the virulence in vivo with the in vitro production of proteinases and phospholipases, hemolytic activity and biofilm development capacity. One culture collection strain of each of seven Candida species (C. albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida metapsilosis, Candida orthopsilosis and Candida parapsilosis) was studied. A double mutant C. elegans AU37 strain (glp-4;sek-1) was infected with Candida by ingestion, and the analysis of nematode survival was performed in liquid medium every 24 h until 120 h. Candida establishes a persistent lethal infection in the C. elegans intestinal tract. C. albicans and C. krusei were the most pathogenic species, whereas C. dubliniensis infection showed the lowest mortality. C. albicans was the only species with phospholipase activity, was the greatest producer of aspartyl proteinase and had a higher hemolytic activity. C. albicans and C. krusei caused higher mortality than the rest of the Candida species studied in the C. elegans model of candidiasis.

Enzymatic characterization of clinical and environmental Cryptococcus neoformans strains isolated in Italy.

BACKGROUND: Cryptococcus neoformans is an encapsulated yeast causing mainly opportunistic infections. The virulence factors involved in cryptococcosis pathogenesis include the presence and the size of the polysaccharide capsule, the production of melanin by phenoloxidase, the growth at 37°C and the enzyme secretion like proteinase, phospholipase and urease. Many other enzymes are secreted by C. neoformans but their role in the fungus virulence is not yet known. AIMS: In order to investigate this topic, we compared the phospholipase production between strains from patients and from bird droppings, and we examined its relationship to phenoloxidase production. We further characterized the strains by determining the activity of 19 different extracellular enzymes. METHODS: Two hundred and five Italian C. neoformans clinical isolates and 32 environmental isolates were tested. Phenoloxidase production was determined by the development of brown colonies on Staib's agar. Extracellular phospholipase activity was performed using the semiquantitative egg-yolk plate method. API ZYM commercial kit was used to observe the production and the activity of 19 different extracellular enzymes. RESULTS: Statistical analysis of the results showed a significantly higher phospholipase activity in the clinical isolates than in the environmental isolates. No significant difference about the phenoloxidase production between both groups was found. Regarding the 19 extracellular enzymes tested using the API ZYM commercial kit, acid phosphatase showed the highest enzymatic activity in both groups. Concerning the enzyme α-glucosidase, the clinical isolates presented a significantly higher positivity percentage than the environmental isolates. A hundred percent positivity in the enzyme leucine arylamidase production was observed in both groups, but the clinical isolates metabolized a significantly greater amount of substrate. CONCLUSIONS: The higher phospholipase production in the clinical isolates group confirms the possible role of this enzyme in the cryptococcosis pathogenesis. The extracellular activities of the enzymes acid phosphatase, α-glucosidase and leucine arylamidase, tested by means of the API ZYM commercial kit, appear to be very interesting. Many studies indicate that these enzymes are involved in the virulence of bacteria and parasites; our results suggest their possible role as virulence factors in Cryptococcus infections too.

Comparison of enzymatic activities in different Candida species isolated from women with vulvovaginitis.

INTRODUCTION: Comparing the activities of secreted enzymes in different fungal species can improve our understanding of their pathogenic role. Secretion of various enzymes by Candida species has been considered for determination of their virulence in different Candida infections including vulvovaginitis. AIMS: The aim of this study was to determine and compare the activity of secreted enzymes in Candidia strains isolated from women suspected to vulvovaginal candidiasis (VVC) and referred to some health centers in Khuzestan, Southwestern Iran. PATIENTS AND METHODS: The vaginal secretion samples were taken by swap from 250 suspected women with symptoms of vulvovaginal candidiasis and cultured on CHROMagar Candida medium. Identification of the isolated Candida from culture positive samples performed by the color of colonies and some standard mycological procedures. Activities of phospholipase, hemolysin-α, hemolysin-ß, esterase and proteinase were measured in vitro by standard laboratory protocols. The enzymatic activity index (EAI) was calculated for each enzyme in accordance to relevant protocols. RESULTS: Totally in eighty cases (32%), a Candida strain was isolated which found to be as 52 (65%) Candida albicans; 12 (15%) C. glabrata; 10 (12.5%) C. dubliniensis; 4 (5%) C. krusei, C. tropicalis and C. parapsilosis species (each=1; 1.3%). Among C. albicans strains, 89.1% produced all studied enzymes, while 86% of C. glabrata strains failed to produce proteinase and phospholipase. The EAIs in decreasing order were as hemolysin-ß=0.2895, hemolysin-α=0.5420, esterase=0.5753, proteinase=0.7413, and phospholipase=0.7446, respectively. Activity of phospholipase, esterase and proteinase secreted by C. albicans and C. dubliniensis were significantly more than those released by C. glabrata and C. krusei, while 86% of C. glabrata strains did not show esterase activity. On the other hand, the activity rates of hemolysin α and ß among all studied isolates were almost similar. CONCLUSION: In the present study, the prevalence of VVC among investigated women was higher than the previous report from Khuzestan but C. albicans has yet remained the predominant agent of VVC in this area. Given to the EAI, the virulence of C. albicans in VVC can be mediated by phospholipase, esterase and proteinases.

ß-Endorphin enhances the phospholipase activity of the dandruff causing fungi Malassezia globosa and Malassezia restricta.

ß-Endorphin is known to stimulate phospholipase production by Malassezia pachydermatis during canine dermatoses. The role of ß-endorphin in Malassezia infection in humans is not well studied. The present study compares the influence of ß-endorphin on Malassezia globosa and Malassezia restricta isolated from patients with seborrhoeic dermatitis/dandruff (SD/D) and healthy controls. Malassezia isolates (five each of the two species from patients and healthy controls) were grown on modified Dixon's agar with or without 100 nmol/L ß-endorphin. Phospholipase activity was quantified based on its ability to hydrolyze L-α-phosphatidylcholine dimyristoyl (phospholipid substrate). Free fatty acid was measured by a colorimetry method. In isolates from patients, the phospholipase activity significantly increased after exposure to ß-endorphin (M. globosa, P = .04; M. restricta, P = .001), which did not occur in isolates from healthy controls. Moreover, after ß-endorphin exposure the patient isolates had significantly higher (P = .0004) phospholipase activity compared to the healthy control isolates. The results suggest that isolates of M. globosa and M. restricta from patients may differ from those of healthy humans.

Virulence Attributes and Antifungal Susceptibility Profile of Opportunistic Fungi Isolated from Ophthalmic Infections.

Investigations of both virulence factors and antifungal susceptibility profiles are crucial for understanding the pathogenesis and prognosis of ophthalmic mycoses. In this study, we investigated the in vitro antifungal susceptibility of amphotericin B (AMB), voriconazole (VRC), and natamycin (NAT) against a set of 50 fungal isolates obtained from patients with ocular mycoses using the Clinical and Laboratory Standards Institute broth microdilution method. In addition, putative virulence factor, such as secretory phospholipases and proteinases, and biofilm formation activity were analyzed. The geometric means (GMs) of the minimum inhibitory concentrations (MICs) of the antifungals across all isolates were the following (in increasing order): VRC (0.70 µg/mL), AMB (0.81 µg/mL), and NAT (1.05 µg/mL). The highest activity against 14 Aspergillus strains was exhibited by VRC (GM MIC: 0.10 µg/mL), followed by AMB and NAT (GM MICs: 0.21 and 0.27 µg/mL), respectively. However, for 12 Fusarium spp., the GM MIC of VRC (2.66) was higher than those of NAT and AMB (GM MICs 1.3 and 0.8 µg/mL, respectively). Proteinase and phospholipase activity were observed in 30 % and 42 % of the isolates, respectively, whereas only 8 % of the isolates were able to produce biofilms. Phospholipase activity was observed in all Fusarium isolates, but not in any of the Aspergillus isolates. In contrast, biofilm-forming capability was detected in 25 % of the Fusarium isolates, but none of the Aspergillus isolates. The differences in the MICs of AMB, VRC, and NAT, biofilm-forming ability and proteinase and phospholipase activities among the isolates were not significant (p > 0.05). Overall, our study suggests no significant correlation between the antifungal susceptibility profiles and virulence attributes of ocular fungal isolates.

Trends in antifungal susceptibility and virulence of Candida spp. from the nasolacrimal duct of horses.

This was a cross-sectional study to investigate the antifungal susceptibility and production of virulence factors in strains of Candida isolated from the outlet and the lumen of the nasolacrimal duct of horses in the state of Ceará, Brazil. The samples were obtained from 103 horses. Sterile cotton swabs were used to collect the material from the outlet of the nasolacrimal duct and urethral probes, for the instillation of 2 ml of saline solution, were used to collect samples from the lumen of the nasolacrimal duct. A total of 77 Candida isolates were obtained, with C. famata, C. tropicalis, C. guilliermondii, and C. parapsilosis sensu lato as the most prevalent species. One isolate (C. glabrata) was resistant to caspofungin. One isolate was resistant only to fluconazole (C. parapsilosis sensu lato), 11 were resistant only to itraconazole (7 C. tropicalis, 2 C. guilliermondii, 1 C. famata, 1 C. parapsilosis sensu lato), while eight C. tropicalis showed resistance to both azoles. Overall, 28 isolates produced phospholipases and 12 produced proteases. These results highlight the importance of investigating the antifungal susceptibility and virulence trends of Candida spp. from the microbiota of the nasolacrimal duct of horses.

Reliability of the agar based method to assess the production of degradative enzymes in clinical isolates of Candida albicans.

The aim of this study was to establish a reproducible protocol using the methodology of hyaline zones around the colonies on specific agar plates for phospholipase and proteinase production. This was an in vitro double-blind experiment, in which the dependent variables were the enzymatic activity measurements (Pz) for the production of phospholipase (Pz-ph) and the production of secreted aspartyl proteinases (Pz-sap). Three independent variables give rise to different measurement protocols. All measurements were carried out at two different moments by four examiners (E1, E2, E3, and E4). The minimum sample size was 30 Candida albicans clinical isolates. Specific agar plates for phospholipase and SAPs production were prepared according the literature. The intra-and inter-examiner reproducibility for each protocol was estimated using the Intraclass Correlation Coefficient (ICC) and its confidence interval (95% CI). Based on the results obtained for both phospholipase and SAPs, there appears to be no consensus on the protocol chosen for each particular examiner. Measuring the colonies in triplicate may be the main factor associated with the increase in measurement accuracy and should therefore take precedence over measuring only one colony. When only one examiner is responsible for taking measurements, a standard protocol should be put in place and the statistical calibration of this researcher should be done prior to data collection. However, if two or more researchers are involved in the assessment of agar plates, our results suggest that the protocols using software to undertake plate reading is preferred.

Phospholipase and Aspartyl Proteinase Activities of Candida Species Causing Vulvovaginal Candidiasis in Patients with Type 2 Diabetes Mellitus.

Few research had investigated the secretion of phospholipase and aspartyl proteinase from Candida spp. causing infection in females with type 2 diabetes mellitus. This research aimed to investigate the prevalence of vulvovaginal candidiasis (VVC) in diabetic versus non-diabetic women and compare the ability of identified Candida isolates to secrete phospholipases and aspartyl proteinases with characterization of their genetic profile. The study included 80 females with type 2 diabetes mellitus and 100 non-diabetic females within the child-bearing period. Candida strains were isolated and identified by conventional microbiological methods and by API Candida. The isolates were screened for their extracellular phospholipase and proteinase activities by culturing them on egg yolk and bovine serum albumin media, respectively. Detection of aspartyl proteinase genes (SAP1 to SAP8) and phospholipase genes (PLB1, PLB2) were performed by multiplex polymerase chain reaction. Our results indicated that vaginal candidiasis was significantly higher among the diabetic group versus nondiabetic group (50% versus 20%, respectively) (p = 0.004). C. albicans was the most prevalent species followed by C. glabrata in both groups. No significant association between diabetes mellitus and phospholipase activities was detected (p = 0.262), whereas high significant proteinase activities exhibited by Candida isolated from diabetic females were found (82.5%) (p = 0.000). Non-significant associations between any of the tested proteinase or phospholipase genes and diabetes mellitus were detected (p > 0.05). In conclusion, it is noticed that the incidence of C. glabrata causing VVC is increased. The higher prevalence of vaginal candidiasis among diabetics could be related to the increased aspartyl proteinase production in this group of patients.

Histone deacetylases inhibitors effects on Cryptococcus neoformans major virulence phenotypes.

Cryptococcus neoformans undergoes phenotypical changes during host infection in order to promote persistence and survival. Studies have demonstrated that such adaptations require alterations in gene transcription networks by distinct mechanisms. Drugs such as the histone deacetylases inhibitors (HDACi) Sodium Butyrate (NaBut) and Trichostatin A (TSA) can alter the chromatin conformation and have been used to modulate epigenetic states in the treatment of diseases such as cancer. In this work, we have studied the effect of NaBut and TSA on the expression of C. neoformans major virulence phenotypes and on the survival rate of an animal model infected with drugs-treated yeasts. Both drugs affected fungal growth at 37°C more intensely than at 30°C; nonetheless, drugs did not affect cell viability at the concentrations we studied. HDACi also provoked the reduction of the fungal capsule expansion. Phospholipases enzyme activity decreased; mating process and melanin synthesis were also affected by both inhibitors. NaBut led to an increase in the population of cells in G2/M. Treated yeast cells, which were washed in order to remove the drugs from the culture medium prior to the inoculation in the Galleria mellonela infection model, did not cause significant difference at the host survival curve when compared to non-treated cells. Overall, NaBut effects on the impairment of C. neoformans main virulence factors were more intense and stable than the TSA effects.

Itraconazole-resistant Candida auris with phospholipase, proteinase and hemolysin activity from a case of vulvovaginitis.

Since the emergence of pathogenic non-albicans Candida species, a number of new isolates have been added to the list. One such unusual species is Candida auris (C. auris), recently isolated and studied in few reports. In this study, a case of vulvovaginitis caused by Candida auris incidentally identified by molecular methods using internal transcribed spacer polymerase chain reaction (ITS PCR) is described. Antifungal susceptibility testing revealed the isolate to be resistant to itraconazole (MIC ≥ 2 µg/ml) and expressed important virulence factors including phospholipase, proteinase and hemolysin activity. The patient was successfully treated with oral fluconazole and did not have any invasive fungemia. Very few cases of this emerging pathogen have been reported. However, its isolation from clinical specimens reveals the significance of non-albicans candida species over C. albicans and the diversity of Candida spp causing infections.

Detection of phospholipase activity of Candida albicans and non albicans isolated from women of reproductive age with vulvovaginal candidiasis in rural area.

BACKGROUND: Vulvovaginal candidiasis (VVC) is most common accounting for 17 to 39% of symptomatic women. Both Candida albicans and non albicans Candida species are involved in VVC. Amongst various virulence factors proposed for Candida, extracellular phospholipases is one of the virulence factor implicated in its pathogenicity. With this background the present study was carried out to find the prevalence of different Candida species and to detect phospholipase producing strains isolated from symptomatic women with VVC. MATERIALS AND METHODS: At least two vaginal swabs from 156 women of reproductive age with abnormal vaginal discharge were collected. Direct microscopy and Gram's stained smear examined for presence of budding yeast and pseudo mycelia followed by isolation and identification of Candida species. Extracellular phospholipase activity was studied by inoculating all isolates on Sabouraud's dextrose egg yolk agar (SDA) medium. RESULTS: Of the 156 women with curdy white discharge alone or in combination with other signs, 59 (37.82%) women showed laboratory evidence of VVC. A total of 31 (52.54%) women had curdy white discharge followed by 12 (20.33%) with other signs and symptoms. C. albicans (62.59%) and non albicans Candida (37.28%) in a ratio of 1.68:1 were isolated. Of the 37 strains of C. albians 30 (81.08%) showed the enzyme activity. Seventeen (56.66%) strains showed higher Pz value of < 0.70 (++++). CONCLUSION: Although there may be typical clinical presentation of Candidiasis. all the patients did not show laboratory evidence of infection. Pregnancy was found to be major risk factor for development of VVC. C. albicans was prevalent species but non albicans species were also frequently isolated. Extracellular phospholipase activity was seen in C. albicans and not in non albicans Candida isolates.

Comparative proteome analysis reveals four novel polyhydroxybutyrate (PHB) granule-associated proteins in Ralstonia eutropha H16.

Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative α/ß-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism.

Protease and phospholipase activities of Candida spp. isolated from cutaneous candidiasis.

BACKGROUND: Cases of superficial and invasive mycoses caused by emerging species of Candida have been increasingly reported over the last thirty years. The production of hydrolytic enzymes plays a central role in the fungal infective process. In Candida infections the secretion of both proteases and phospholipases are well-known virulence attributes. AIMS: To determine the protease and phospholipase production from 58 human clinical isolates of Candida obtained from individuals with cutaneous candidiasis seen in the Human and Veterinary Diagnostic Mycology Sector from Universidade Federal Fluminense (UFF), Brazil, from November 2008 to August 2009. METHODS: Fungal identification was performed using biochemical tests. Proteolytic activity was detected on agar plates containing bovine serum albumin, and phospholipase production was determined on egg-yolk plates. RESULTS: The Candida species isolated were Candida parapsilosis (27.59%), Candida famata (18.96%), Candida albicans (15.52%), Candida haemulonii (12.06%), Candida ciferri (8.62%), Candida guilliermondii (6.90%), Candida tropicalis (5.17%) and Candida lipolytica (5.17%). All isolates of C. albicans produced both protease and phospholipase. As regards the isolates of non-C. albicans Candida species, 53.06% and 4.08% were able to produce protease and phospholipase, respectively. For example, the majority of isolates of C. parapsilosis (15/16) produced protease, while 40% of C. ciferri isolates (2/5) were phospholipase producers. This study shows, for the first time, that C. ciferri and C. haemulonii strains were able to produce protease. CONCLUSIONS: Collectively, our results showed that different species of Candida isolated from cutaneous lesions were able to produce proteases and/or phospholipases, which are multifunctional molecules directly involved in the infectious process of these fungi.