Production Of Extracellular Hydrolytic Enzymes Research Articles

Assessing variability among culturable phylloplane basidiomycetous yeasts from Italian agroecosystems

This study analysed basidiomycetous yeasts isolated from the phylloplane of crops and spontaneous plants in Italian agroecosystems. A total of 25 species belonging to 17 genera were recognized by analysing 83 isolates from vineyards and orchards, that are not treated with synthetic fungicides, and adjacent natural areas. Rhodotorula graminis and Filobasidium magnum were the most frequent species but 13 others were represented by a single isolate (e.g., Buckleyzyma salicina, Pseudozyma prolifica, and Moniliella megachiliensis). Preliminary analysis of (GTG)5-PCR fingerprinting revealed high genetic intraspecific heterogeneity. All isolates were characterized by their production of extracellular hydrolytic enzymes and their sensitivity to six commercial fungicides used in Italy. The isolates displayed great variability in these phenotypic traits, which play an important role in the survival of yeast populations in agroecosystems. Most of them exhibited lipolytic, proteolytic, β-glucosidase and pectinolytic activities, but only three (F. magnum, Kwoniella mangroviensis and Ps. prolifica) also had cellulolytic and amylolytic activity. Most isolates were sensitive to four fungicides, and one R. graminis isolate was resistant to all six. This heterogeneity was not related to the geographical origin of the isolates. The lack of selective factors (i.e. pesticide treatments) in the sampling fields and the presence of adjacent natural areas may have favored the maintenance of an elevated level of strain diversity. This study provides new information on phylloplane basidiomycetous yeasts in agroecosystems and opens the way to further investigations into the impact of agricultural practices on the microbial diversity of these natural habitats.

Assessment of Proteinase and Phospholipases Enzymes Isolated from Pathogenic Candida species from Women Attending Antenatal Clinic at Mbeya Zonal Referral Hospital, Southern Highland Regions of Tanzania

Fungi are a global cause of vaginal infections, with vaginal candidiasis largely afflicting tropical regions including most parts of sub-Saharan Africa. In Tanzania, baseline studies have established the prevalence of Candida species among symptomatic and asymptomatic pregnant women in Dar es Salaam (Namkinga et al., 2005; Namkinga 2012) and in Mwanza (Mushi et al., 2019). However, no study that has been done to determine the above-mentioned enzymes form Candida species in Southern Highland Regions of Tanzania. Several factors have been reported to contribute to the virulence and pathogenicity of Candida, among others the production of extracellular hydrolytic enzymes, particularly phospholipase and proteinase. Therefore, this study was designed to investigate the in vitro production of phospholipases and proteinases enzymes from isolated pathogenic Candida spp from pregnant women.Of the 280 samples collected from symptomatic and asymptomatic pregnant women attending antenatal clinics at Mbeya Zonal Referral Hospital were tested for several microbiological methods such as the wet mount microscopic preparations (hanging drop) and the Gram stained smears for microscopy, cultures on Sabouraud’s dextrose agar and on Conidia enhancing medium (Corn meal agar), germ tube test, biochemical tests, sugar fermentation-assimilation tests and molecular test to characterize Candida species.Phospholipase production was performed in egg yolk medium while the production of proteinase was done in a medium containing bovine serum albumin. All analyses were performed in triplicates. The results showed that; out of the 280 tested, 155 (74.3%) isolates were phospholipase positive while 201 (96.6%) were positive for proteinase activity. C. albicans was the species with the highest number of positive isolates for proteinase and phospholipase 110 (95.6%), and most strains of C. albicans produce both enzymes (phospholipases and proteinases). The non-albicans Candida isolates were also producers of hydrolytic enzymes that, consequently, might be able to cause infections as favorable conditions arises.

Screening and characterization of thermostable enzyme-producing bacteria from selected hot springs of Ethiopia.

Thermostable microbial enzymes play an important role in industries due to their stability under harsh environmental conditions, including extreme temperatures. Despite their huge application in different industries, however, the thermostable enzymes of thermophilic microorganism origin have not yet been fully explored in Ethiopia. Here, we explored thermophilic bacteria and their enzymes from selected hot spring water and sediment samples. Accordingly, thermophilic bacteria were isolated and screened for the production of extracellular hydrolytic enzymes. Promising numbers of isolates were found as producers of the enzymes. The potent enzyme producers were further identified using matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry analysis and 16S rRNA gene sequencing. The findings revealed the presence of potential hydrolytic enzyme-producing thermophilic bacteria in hot springs of Ethiopia and necessitate further comprehensive study involving other extreme environments. Our findings also revealed the potential of Ethiopian hot springs in the production of thermostable enzymes of significant application in different industries, including food industries.

Study of Endophytes as Biocontrol Agents Vis-a-Vis their Compatibility to Fungicides in Grapes

The present study was conducted at ICAR-National Research Centre for Grapes in Pune, Maharashtra, India during 2021–2022 to study endophytic bacteria as biocontrol agents vis-a-via their compatibility to fungicides in grapes. Eighteen endophytic bacteria were isolated from nine varieties of grapevine (Vitis vinifera L.) viz. Manjari Naveen (MN), Nanasaheb Purple (NP), Thompson Seedless (TS), Crimson Seedless (CS), Manik Chaman (MC), Maruti Seedless (MS), Fantasy Seedless (FS) and 2A Clone (2A). The distinct colonies were selected, sub-cultured, purified and subjected to analysis of macroscopic and microscopic features followed by screening of bacterial isolates for plant growth-promoting (PGP) traits, extracellular hydrolytic enzyme production, and abiotic stress tolerance assay. Eighteen diverse endophytic bacteria were found to be Gram positive to Gram negative and with white, whitish yellow and whitish red colonies. Different biochemical tests which were needful for the identification of the bacteria were performed. Antibiotic sensitivity and antifungal assays were conducted with five antibiotics and pathogenic fungi Colletotrichum gloeosporioides respectively. Results of dual culture plate assay for antimicrobial activity revealed that 10 isolates showed significant growth inhibition of the test pathogen and were selected for the compatibility studies. The results highlighted that most of the isolates were highly compatible with all the fungicides used for powdery mildew, downy mildew, anthracnose and bacterial leaf spot. The work indicated the importance of the endophytic bacteria which can be used as promising biocontrol agents for grapevine disease management.

A comparative study on the production of extracellular hydrolytic enzymes of C. albicans and non-albicans Candida species isolated from HIV+/AIDS patients and healthy individuals.

Oropharyngeal candidiasis is the most prevalent opportunistic fungal infection in patients with human immunodeficiency virus (HIV) as well as other immunodeficiency disorders, which is caused by various Candida species, mostly Candida albicans. Studies have shown that Candida isolates differ in their pathogenicity. These variations are attributed to virulence factors, host characteristics, and the target tissue. This study aimed to determine and compare the secretion of hydrolytic enzymes in C. albicans and non-albicans Candida species isolated from HIV+/AIDS patients and healthy individuals. Samples were taken from 201 patients with HIV and 118 healthy individuals. The samples were identified by macroscopic, phenotypic, and molecular methods, and virulence factors were subsequently measured. Statistical differences in enzymatic activity of various Candida isolates were calculated (P<0.0001). In total, 95 samples (47.20%) from patients and 46 samples (38.90%) from healthy individuals were positive for the growth of different Candida species. There were 39 (41.10%) and 36 (78.30%) C. albicans in patients and healthy individuals, respectively, as well as 56 (58.90%) and 10 (21.70%) non-albicans species in patients and healthy subjects, respectively. All the enzymes produced by Candida species enzymes were at low, medium, and high levels. Hemolysin activity in Candida species isolated from patients was significantly higher, compared to healthy individuals. Moreover, the activity of all C. albicans enzymes in patients was significantly higher than other Candida species. The C. albicans isolated from HIV-positive individuals secreted higher amounts of exoenzymes, and can cause oropharyngeal candidiasis and become a source of candidiasis for the host.

Organic Matter Degradation in Energy-Limited Subsurface Environments—A Bioenergetics-Informed Modeling Approach

Microbial degradation of organic matter is a key driver of subsurface biogeochemistry. Here, we present a bioenergetics-informed kinetic model for the anaerobic degradation of macromolecular organic matter that accounts for extracellular hydrolysis, fermentation, and respiration. The catabolic energy generated by fermentation and respiration is allocated to biomass growth, production of extracellular hydrolytic enzymes, and cellular maintenance. Microbial cells are assumed to exist in active or dormant states with marked differences in maintenance energy requirements. Dormant cells are further assumed to fulfill their maintenance energy requirements by utilizing their own biomass instead of relying on external substrates. When the catabolic Gibbs energy production for a given functional group of microorganisms exceeds the total maintenance energy requirement of the active cells, biomass growth, re-activation of dormant cells, and production of extracellular hydrolytic enzymes are possible. The latter, in turn, allows the microbial community to access more of the available external organic substrates. In the opposite case, active cells decay or become dormant. We apply the model to simulate the anaerobic degradation of cellulose by a hypothetical microbial community consisting of cellulolytic fermenting bacteria and sulfate-reducing bacteria, under conditions representative of those encountered in water-saturated subsurface environments.

Quorum sensing disruption regulates hydrolytic enzyme and biofilm production in estuarine bacteria.

Biofilms of heterotrophic bacteria cover organic matter aggregates and constitute hotspots of mineralization, primarily acting through extracellular hydrolytic enzyme production. Nevertheless, regulation of both biofilm and hydrolytic enzyme synthesis remains poorly investigated, especially in estuarine ecosystems. In this study, various bioassays, mass spectrometry and genomics approaches were combined to test the possible involvement of quorum sensing (QS) in these mechanisms. QS is a bacterial cell-cell communication system that relies notably on the emission of N-acylhomoserine lactones (AHLs). In our estuarine bacterial collection, we found that 28 strains (9%), mainly Vibrio, Pseudomonas and Acinetobacter isolates, produced at least 14 different types of AHLs encoded by various luxI genes. We then inhibited the AHL QS circuits of those 28 strains using a broad-spectrum lactonase preparation and tested whether biofilm production as well as β-glucosidase and leucine-aminopeptidase activities were impacted. Interestingly, we recorded contrasted responses, as biofilm production, dissolved and cell-bound β-glucosidase and leucine-aminopeptidase activities significantly increased in 4%-68% of strains but decreased in 0%-21% of strains. These findings highlight the key role of AHL-based QS in estuarine bacterial physiology and ultimately on biogeochemical cycles. They also point out the complexity of QS regulations within natural microbial assemblages.

Los aislados de hongos del género Trichoderma inducen la resistencia al marchitamiento del guisante causada por Fusarium oxysporum f. sp. pisi a través de la inhibición competitiva

Four different Trichoderma species were isolated from the soil rhizosphere of pea field from selected areas of Punjab, Pakistan. Based on morphological and cultural characteristics species were recognized as T. harzianum, T, viridae, T. hamatum and T. koningii. The isolated species were screened for the production of extracellular hydrolytic enzymes. The strains were found positive for chitinase and glucanase activities.&nbsp; Fusarium oxysporum f. sp. pisi was isolated from roots of wilted pea plants. In-vitro biocontrol potential of the isolated Trichoderma species was assayed by dual culture technique against Fusarium oxysporum f. sp pisi. The maximum growth inhibition was observed by T. viridae (76.42%) followed by T. harzianum (74.29%), T. koningii (71.43%) and T. hamatum (69.64%), after 7 days of incubation. All four isolated species of Trichoderma were used in confrontational assay against Fusarium oxysporum pisi (FOP). A competition of Trichoderma sp. v/s FOP was evident, all Trichoderma strains showed strong antagonistic activity that was clearly observed on dual culture agar plates and further confirmed under scanning electron microscopy (SEM). Pot experiments also confirmed a very strong competitive inhibition of FOP evidently due to presence of glucanase and chitinases in Trichoderma spp. The primary screening and basic findings of present work will be helpful to obtain an efficient and novel biocontrol agent for further experimental trials on pea plants and may also enhance the chance of using Trichoderma species in integrated disease management (IDM) programs as an effective biological agent against several phyto-pathogens.

Phosphorus deficiencies invoke optimal allocation of exoenzymes by ectomycorrhizas

Ectomycorrhizal (EM) fungi can acquire phosphorus (P) through the production of extracellular hydrolytic enzymes (exoenzymes), but it is unclear as to the manner and extent native EM fungal communities respond to declining soil P availability. We examined the activity of six exoenzymes (xylosidase, N-acetyl glucosaminidase, β-glucosidase, acid phosphomonoesterase, acid phosphodiesterase [APD], laccase) from EM roots of Pseudotsuga menzesii across a soil podzolization gradient of coastal British Columbia. We found that APD activity increased fourfold in a curvilinear association with declining inorganic P. Exoenzyme activity was not related to organic P content, but at a finer resolution using 31P-NMR, there was a strong positive relationship between APD activity and the ratio of phosphodiesters to orthophosphate of surface organic horizons (forest floors). Substantial increases (two- to fivefold) in most exoenzymes were aligned with declining foliar P concentrations of P. menzesii, but responses were statistically better in relation to foliar nitrogen (N):P ratios. EM fungal species with consistently high production of key exoenzymes were exclusive to Podzol plots. Phosphorus deficiencies in relation to N limitations may provide the best predictor of exoenzyme investment, reflecting an optimal allocation strategy for EM fungi. Resource constraints contribute to species turnover and the assembly of distinct, well-adapted EM fungal communities.

A comparative study on production of extracellular hydrolytic enzymes of Candida species isolated from patients with surgical site infection and from healthy individuals and their co-relation with antifungal drug resistance

BackgroundSurgical site infection (SSI) is a crucial dilemma of surgery. Patients with SSIs not only face difficulty in treatment but also bear extra cost with high mortality rate. Resistant strains of Candida have emerged as an important nosocomial pathogen. Proteinase and phospholipase are exo- enzymes of Candida species, have importance with respect to their contribution in diseases. This study focused on prevalence of Candida species in surgical wound, their resistance to antifungal drugs, co-relation of these resistance with virulence potential of Candida species and comparison of production level of exo-enzymes of Candida species isolated from patients with SSIs and healthy individuals to highlights their role in SSIs.ResultsA total of (n = 555) swab samples were investigated. (n = 450) samples were collected from patients with SSIs and (n = 105) were collected from healthy individuals. Samples were subjected for the identification of Candida species which were subsequently investigated for antifungal susceptibility, MICs and enzymatic activity of Candida species. Out of 128 strains of Candida spp. isolated from SSIs, 54(42.18%) were identified as C. albicans followed by C. glabrata 32(25%), C. parapsilosis 17(13.28%), C. krusei 13(10.16%) and C. tropicalis 12(9.38%). C. albicans isolates showed 100% susceptibility to voriconazole and amphotericin B followed by itraconazole 98% and fluconazole 89%. Out of 6 fluconazole resistant C. albicans 5(83.33%) were able to produce phospholipase while out of 48 fluconazole-susceptible strains 17(35.42%) were found to be phospholipase producer. Out of 54 C. albicans isolated from surgical wound 46(85.18%) and 49(90.74%) were found to be phospholipase and proteinase producer respectively, whereas out of 20 C. albicans isolates from healthy subjects 14(70%) produce proteinase and 12(60%) produce phospholipase. There were significant statistical differences found between the level of enzyme production by C. albicans, in relation to both sites (P = 0.014).ConclusionStudy revealed that prevalence of Candida species is high in SSIs. Phospholipase and proteinase activity were more pronounced in Candida Species from surgical wound in contrast to species from healthy individuals suggests these enzymes may have been responsible for the severity of infection in surgical wound patients.

Effect of Heterodera schachtii female age on susceptibility to three fungal hyperparasites in the genus Hyalorbilia.

Three closely related nematophagous fungi in the genus Hyalorbilia were compared for their ability to parasitize females and eggs of Heterodera schachtii at different developmental stages. DoUCR50, StM, and ARF were originally isolated from Heterodera schachtii, Meloidogyne incognita, and Heterodera glycines, respectively. Phylogenetic analysis and pairwise sequence analysis showed that DoUCR50 and StM are more closely related to each other than they are to ARF. DoUCR50 parasitism suppressed 100% of the J2 hatch from 3-week-old H. schachtii females and 75% of the hatch from 4-week-old females. Eggs within 5-week-old females were resistant to parasitism, and hatch of J2 was unaffected by exposure to DoUCR50. StM and ARF did not reduce the hatch of J2 from H. schachtii females of any age. Eggs removed from females and spread onto water agar cultures of the fungi were mostly resistant to parasitism. DoUCR50 parasitized only 16% of such eggs from 3-week-old females. Extracellular hydrolytic enzyme production by the three fungal strains grown on PDA or parasitized H. schachtii females was evaluated using API ZYM (bioMérieux) test strips. All three fungi produced extracellular hydrolytic enzymes when grown on PDA or H. schachtii females. Trypsin-like protease activity was uniquely detected in DoUCR50 grown on PDA and H. schachtii females, with the highest activity associated with the fungus grown on parasitized females.

Assessment of Extra Cellular Enzymes of Bacteria Isolated from Mangrove Rhizosphere Soil of Different Places of Gujarat in Monsoon Season

Background/objectives: Mangroves are one of the most diverse ecosystems on the earth and harbours many organisms, including extremophiles which help the ecosystem to grow and survive despite harsh environment. These microbes produce novel enzymes as well as secondary metabolites that find major applications in extreme environments. Methodology: In this study, total 56 bacteria were isolated from rhizospheric soil of mangrove collected from seven sampling sites of Gujarat, India i.e. Kandla, Mundra, Tuna, Guneri, Machhad, Diu, and Madhwad. These isolates were screened for their production of extracellular hydrolytic enzymes i.e. amylase, protease, cellulase, and catalase. All the isolates were also assessed for their ability to survive in different salt concentration ranging from 0.5% to 25% in Zobell marine agar. Findings: After 48 h of incubation, isolates K10–2(11)ZO and M100(5)NASW had the highest amylase activity, K100(1)NASW had the highest cellulase activity, and G10–2(9)ZO had the highest protease activity. Among all sites, Madhwad had the highest catalase positives. Three isolates named as MD10–2C3, MW10–3C2, and MWC4 emerged as possible extreme halotolerant and were able to grow at 25% of salt concentration while Diu10–4C1, MW10–3, and G10–1(5) ZO were also possible halotolerant and were able grow in till 20% of salt concentration. Application/ improvements: Few isolates that had showed higher extracellular enzyme activity and could be potential high producers that can be used in industries such as detergent industry, textile industries, environmental remediation, etc.Keywords: Mangroves, Enzymes, Cellulase, Amylase, Protease, Extreme Halotolerant

Some characteristics of phosphate solubilizing rhizobacteria as an ecological strategy for sustainable agriculture

Abstract With the aim to select rhizobacteria exhibiting several plant growth promoting traits, 5 isolates were evaluated. Phosphate (P)solubilization halos were between 0.4 and 0.9 cm, and P concentrations ranged between 66.83 and 339.39 mg l-1, accompanied by media pH decrease. Three isolates, PP14, PP17, and RL90, synthesized indole acetic acid, while PE66 and PE85 could hydrolyze ACC. All isolates produced siderophores and hydrogen cyanide except RL90 and PP17. For extracellular hydrolytic enzymes production, RL90 was positive for cellulase and protease. All isolates suppressed at less one phytopathogenic fungus growth, while the best bacterial phytopathogens inhibition was obtained for PE85.

Characterization of thermophilic fungi producing extracellular lignocellulolytic enzymes for lignocellulosic hydrolysis under solid-state fermentation

BackgroundThermotolerant lignocellulolytic enzymes have become a subject of interest in industrial processes due to their ability to degrade lignocellulosic polysaccharides. Development of cost-effective, large-scale screening for production of desirable enzymes by thermophilic fungi is a challenge. The present investigation focused on isolating, screening, and identifying industrially relevant thermophilic producers of lignocellulolytic enzymes from various locations in the Warangal district, Telangana, India.ResultsFifteen thermophilic fungi were isolated from soil on their ability to grow at 50 °C and were screened for their activity of cellulase, hemicellulase, and lignin degradation based on holo zone around colonies. The appearance of the black color zone of diffusion in esculin agar is a positive indication for the β-glucosidases activity test. Out of fifteen isolates, Aspergillus fumigatus JCM 10253 have shown as a potential producer of extracellular enzymes for lignocelluloses degradation showing higher activity for cellulase (EI 1.50) as well as β-glucosidase (4 mg/mL), simultaneously for xylanase (EI 1.18) by plate assay methods. A. fumigatus JCM 10253 was selected for extracellular hydrolytic enzymes production under solid-state fermentation. Maximum CMCase (26.2 IU/mL), FPase (18.2 IU/mL), β-glucosidase (0.87 IU/mL), and xylanase (2.6 IU/mL) activities were obtained after incubation time of 144 h at 50 °C. The thermostability of crude cellulase showed the optimum activity at 60 °C and for FPase, β-glucosidase, and xylanase at 50 °C which recommended that the enzymes have a potentially significant role in the biofuel industries.ConclusionThe high titer production of active enzymes that cleave different β-1,4-glycosidic bonds still remains a challenge and is the major bottleneck for the lignocellulosic conversion. In particular, the finding of thermostable enzymes which would allow the development of more robust processes is a major goal in this field.

Determination of Extracellular Hydrolytic Enzyme Production Capabilities of Acidithiobacillus Bacteria Isolated from Awasppi River in Sulaymaniyah, Iraq and 16S rDNA Analysis

Bu arastirmada, Kuzey Irak'in Suleymaniye sehrinde bulunan Awasppi nehrinden ve Sangaw'daki Darzilais sulfur kaynakli magaradan alinan su ve camur orneklerinden izole edilen Acidithiobacillus bakterilerinin bazi hidrolitik ektraseluler enzim aktiviteleri incelenerek, 16S rDNA gen bolgelerinin sekans analizi temeline dayali filogenetik pozisyonlari belirlenmistir. Izolatlarin katalaz aktivitesi pozitif iken, lipaz, proteaz, ksilanaz ve amilaz aktivitesi negatif olarak belirlenmistir. Izolatlarin Genomik DNA’lari izole edilerek 16S rDNA gen bolgesi PZR amplifikasyonu ile gerceklestirilmistir. Turler arasindaki uzakligi belirlemek icin Maximum Likelihood algoritmasi kullanilarak olusturulan filogenetik agac NCBI’dan elde edilmis Acidithiobacillus turlerine ait gen dizileri ve calismamizda kullandigimiz 2 izolat guclu bir homoloji ile kumelendigi gozlemlenerek genetik pozisyonlari ortaya konmustur. Karsilastirmali 16S rDNA gen dizisi analizi sonucunda, G1 izolatinin Acidithiobacillus thiooxidan s’a % 99 benzerlik gosterdigi ve G2 izolatinin Acidithiobacillus ferrooxidans ’a % 99 benzerlik gosterdigi belirlenmistir

Prevalence of non-albicans Candida species versus Candida albicans in critical care patients of a tertiary care hospital

Introduction: Candida species are the most common cause of opportunistic fungal infections. Although C.albicans is most common cause of candidiasis, a shift towards non-albicans Candida species is evident in recent years. The transition of Candida spp. from commensal to a potent pathogen is facilitated by a number of virulence factors viz. adherence to host tissues and medical devices, biofilm formation, and secretion of extracellular hydrolytic enzymes. Objective: To study the prevalence of C.albicans &amp; Non-albicans isolates in critical care settings and determine their virulence factors and antifungal susceptibility profile. Material &amp; Methods: The present study was carried out in the Department of Microbiology, SGRIMSAR, Amritsar during the period of July 2014 to June 2016. Candida strains isolated from various clinical samples received from different ICUs of the hospital were included in the study. The isolates were identified upto species level by both conventional and automated methods (vitek 2 compact system) as per CLSI guidelines. Relevant history of all the patients was taken. They were also screened for the production of virulence factors such as biofilm formation, haemolytic activity, and production of extracellular hydrolytic enzymes i.e. coagulase. Results: Out of the 115 isolates obtained from various ICUs, most common isolate was C. tropicalis 60/115(52.17%) followed by C. albicans 45/115(39.13%), C.utilis 7/115(6.25%). Isolates of C. lusitaniae, C. parasillosis &amp; C. glabrata were 1/115 (0.86%) each. Among these 95/115(82.6%), 75/115(65.2%), and 83/115(72.17%) isolates showed biofilm formation, coagulase production and haemolytic properties respectively. Conclusions: Our study showed a shift among Candida species from albicans (39.13%) to non-albicans (60.86%), thus stressing their presence as major fungal pathogens in critical care settings.

In vitro study of biological activity of four strains of Burkholderia gladioli pv. agaricicola and identification of their bioactive metabolites using GC-MS.

This research was carried out to study in vitro antibacterial activity of 4 strains of Burkholderia gladioli pv. agaricicola (Bga) against G+ve Bacillus megaterium and G−ve Escherichia coli, haemolytic activity against the cell membrane of erythrocytes, the production of extracellular hydrolytic enzymes and finally, the pathogenicity against Agaricus bisporus flesh blocks. Chemical structure of bioactive substances of the most bioactive strain (ICMP 11096) was established using gas chromatography–mass spectrometry (GC–MS). All the studied Bga strains inhibited the growth of the two tested bacteria although some growing substrates negatively influenced the antimicrobial substance production. The same Bga strains showed highly haemolytic activity and were able to produce 3 hydrolytic enzymes, i.e. chitinase, glucanase and protease. In pathogenicity assays, the considered Bga strains resulted virulent for A. bisporus. The GC–MS for compounds from Bga ICMP 11096 were compatible with the structure of two bioactive fatty acids identified as methyl stearate and ethanol 2-butoxy phosphate with mass spectrum m/e 298 and 398, respectively.

Evaluation of Virulence Factors of Candida Species Isolated from Superficial versus Systemic Candidiasis

Background: Various virulence factors are contributing to the colonization and pathogenicity of Candida during both superficial and systemic infections Objectives: The current study was conducted to investigate production of extracellular hydrolytic enzymes, hemolysis and biofilm formation among Candida species isolated from superficial versus systemic candidiasis and to compare the production of these virulence factors among albicans and non-albicans Candida species, in addition to comparing hemolysis and hydrolytic enzymes production under aerobic and anaerobic conditions. Methodology: A total of 51 Candida strains isolated from vulvovaginal candidiasis and 44 strains isolated from systemic candidiasis were included in the study. Germ tube test and Hi Crom Candida Differential Agar medium were used for species identification of the isolates. The Candida isolates were tested for expression of hemolytic activity, phospholipase, proteinase and esterase production under aerobic and anaerobic conditions, in addition to their ability of biofilm formation. Results: In both groups, 55 isolates were identified as Candida albicans (C. albicans), while 44 isolates were identified as non-albicans species. Hemolytic activity and biofilm formation were significantly higher among the systemic Candida isolates than superficial isolates, while expression of proteinase activity was significantly higher among superficial isolates. Expression of phospholipase and esterase was significantly higher among C. albicans isolates than non-albicans isolates. Expression of proteinase and esterase activities was significantly better under aerobic conditions than under anaerobic conditions. Conclusions: Hemolytic activity and bioflm formation were more evident among the systemic isolates while proteinase production appears to be more evident among the vaginal isolates. Phospholipase production is almost an exclusive characteristic feature of C. albicans, while C. albicans is the leading species in terms of esterase production. Aerobic conditions favor expression of proteinase and esterase activities.

Extracellular hydrolytic enzymes production of susceptible and fluconazole-resistant Candida albicans after photodynamic therapy

Curcumin (CUR) is a pigment extracted from Curcuma longa L. with great potential as a photosensitizer for photodynamic inactivation of oral biofilms. However its low solubility in aqueous solutions is a challenge. In order to indicate this therapy for clinical use with adequate efficacy and safety, the investigation of biocompatible vehicles able to solubilize the CUR retaining its photodynamic effect is required. In this context, the first step is to investigate the photodynamic behavior of new formulations. This study assessed the photodegradation of CUR at two concentrations (60 and 80 M) prepared in different vehicles (Oleic Acid, Chitosan and Emulgin). Aliquots of each formulation were transferred to 96 well plates and illuminated for 1, 2, 3, 4, 5,10, 15, 20, 25 and 30min with a blue LEDs system ( =440nm, I=0.022mW/cm2). After each illumination time, a measure of the absorbance spectra of the formulations was taken. Photobleaching rates were calculated and it was concluded that, for any formulation tested, the maximum illumination time should be of 15min because after that there will be noCURavailable to continuepromotingphotodynamic effect. Thus, the most stable formulation was the one prepared with chitosan (80 M).

In vitro antagonism of Phytophthora capsici and Fusarium solani by bacterial isolates from Sarawak

Aims: Phytophthora capsici and Fusarium solani are common fungal pathogens causing severe diseases that lead to economic loss in pepper industry, especially in Sarawak. In response to the infections, chemical approach is more common; nevertheless, biological control is more favorable to control fungal pathogens. Biological control approach greatly reduces the problems associated with chemical applications and it restores balance of the natural environment. Here we present the ongoing work to study the action of antagonistic bacteria, Bacillus sp. and Pseudomonas sp., that produce volatile and non-volatile antifungal compounds against P. capsici and F. solani on pepper plants. Methodology and results: A total of seven bacterial candidates were isolated from different locations and tested for their antagonistic properties against P. capsici and F. solani in a dual culture assay and extracellular metabolite test. Extracellular hydrolytic enzymes production was also monitored and followed by genotypic indentification. Preliminary antagonism tests indicated that bacterial isolate Pep3 and Pep4 inhibit up to 50% of the growth of P. capsici and F. solani as compared to the control. Subsequent investigation on extracellular hydrolytic enzyme production revealed that both bacterial isolates are capable of secreting hydrolytic enzymes. Microscopic and genotypic analyses identified the bacterial isolates Pep3 as Bacillus amyloliquefaciens (KJ461444) and Pep4 as Pseudomonas pachastrellae (KM460937). Conclusion, significance and impact of study: B. Amyloliquefaciens (KJ461444) and P. pachastrellae (KM460937) inhibited the growth of P. capsici and F. solani thus reflecting the potential of the produced metabolites to be purified and used in combating plant pathogenic fungi.