Genetic diversity of Cryptosporidium isolates from human populations in an urban area of Northern Tunisia.

International audienceCryptosporidium is an enteric parasite infecting a wide range of hosts. It has emerged as an important cause of chronic life-threatening diarrhea in humans worldwide. Several subtypes of Cryptosporidium sp. have been described to be responsible for several large outbreaks related to water contamination in developed countries. However, there is a lack of information in the genetic diversity of Cryptosporidium among human population especially in developing countries. The present study aimed to update and report the genetic diversity of human Cryptosporidium spp. at the subtype level in an urban area of Tunisia using the 18S rRNA and gp60 gene. Genotyping of 42 Cryptosporidium positive isolates from different human populations at the 18S rRNA locus has identified three Cryptosporidium species: C. hominis (n = 20), C. parvum (n = 19), C. meleagridis (n = 2) and a co-infection C. hominis/C. meleagridis (n = 1). The sub-genotyping of these isolates at the 60-kda glycoprotein (gp60) locus was possible in 40 cases. It showed the presence of three subtype families (IIa, IIb and IIc) within C. parvum, a single subtype family within C. hominis and C. meleagridis isolates (Ia and IIIb respectively). Several subtypes were implicated in different human populations with the dominance of IaA26G1R1, IIaA15G2R1, IIdA16G1R1, IIdA22G2R1 and IIIbA26G1R1 variant respectively for C. hominis, C. parvum and C. meleagridis. The distribution of Cryptosporidium isolates in urban area of Northern Tunisia was dominated by the anthroponotic transmission via C. hominis species and the IIc subtype of C. parvum. However, zoonotic transmission is still possible in this region via zoonotic subtypes of C. parvum (IIa and IId) and C. meleagridis (IIIb). Subtype diversity was higher in this area

Seroprevalence of Toxoplasma gondii infection among horses in Tunisia.

International audienceBACKGROUND: The present study was conducted to investigate the serological survey of Toxoplasma antibodies in local.horses from three major regions: a neighbourhood of a city in the North (Sidi Thabet), a neighbourhood of a city on the coast (Monastir) and a neighbourhood of a city in the middle (Battan) of Tunisia (North of Africa). METHODS: A total of 158 serum samples were obtained from clinically healthy horses which consisted of 111 (32 female, 79 male) 2-10 years old and 47 (11 female, 36 male) older than 10 years. All of the horses were tested for antibodies to T. gondii using the Modified Agglutination Test (MAT). RESULTS: According to MAT results, antibodies to T. gondii were found in 28 (17.7%) of 158 sera with the titers of 1:20 in 20 horses, 1:40 in 1 horse, 1:80 in 2 horses, 1:160 in 2 horses, 1:320 in 1 horse and ≥1:640 in 2 horses. Anti-T. gondii antibodies were found in 18 (16.2%) of 111 horses (2-10 years old) and 10 (21.2%) of 47 horses (older than 10 years old). Six (13.9%) out of 43 female had anti-toxoplasma antibodies and 22 (19.1%) from 115 males remained positive. CONCLUSION: Statistically significant differences in age groups and genders were observed between the seropositive and seronegative horses using the Chi square X(2) test. Other statistical correlation was also reported concerning horse breed

Polymorphism study of Cryptosporidium hominis gp60 subtypes circulating in Tunisia.

International audienceCryptosporidium spp. are a major cause of gastrointestinal diseases in humans worldwide. While a single subtype of Cryptosporidium hominis has been shown to be responsible for several large outbreaks related to water contamination in developed countries, little is known about the epidemiology of C. hominis in developing countries. This study reports the first genetic characterization of C. hominis at the subtype level in several human populations in Tunisia using the gp60 gene. Eighteen isolates were identified as C. hominis by a restriction fragment length polymorphism (RFLP) analysis. The prevalence of this species in different human populations ranges from 1.53% to 13.04% with a high prevalence being reported in immunocompromised children (13.04%) followed by patients with malignent myeloma (5.5%) and HIV-infected patients (4.59%). The gp60 analysis on C. hominis isolates, performed in 14 cases, showed the presence of a single subtype family: "Ia". Different subtypes were identified within this family (A11G1R1, A12R3, A23G1R1, A26G1R1, A27G1R1, A28G1R1). The IaA26G1R1 subtype was the most dominant subtype described in this area (50%). Despite the high genetic diversity of Cryptosporidium spp, a low heterogeneity at the subtype level was observed within C. hominis circulating in Tunisia. This distribution is an indicator for intensive and stable anthroponotic cryptosporidiosis in this region. Besides, the presence of a unique genotype in 5 HIV-infected patients attending the same hospital ward suggests the possible occurrence of hospital-acquired infection and underlines the need to implement preventive measures to avoid nosocomial transmission

Antioxidant, antibacterial, and antileishmanial potential of Micromeria nervosa extracts and molecular mechanism of action of the bioactive compound

Aims: This study aimed to determine the antibacterial and antileishmanial potential of Micromeria nervosa extracts. The identification of the antileishmanial compound and the study of its molecular mechanism of action have also been undertaken. Methods and results: Ethanol extract showed high polyphenol content and diethyl ether extract exhibited high DPPH scavenging and low beta-carotene bleaching activity (IC50 = 13.04 ± 0.99 and 200.18 ± 3.32 μg mL−1 , respectively). However, diethyl ether extract displayed high antibacterial activity against Gram-positive strains including methicillin-resistant Staphylococcus aureus (MIC = 31.25 μg mL−1 ), Staph. aureus ATCC6538 (MIC = 62.5 μg mL−1 ), and Listeria monocytogenes ATCC 19115 (MIC = 125 μg mL−1 ), as well as high antileishmanial activity against the promastigote forms of L. infantum and L. major (IC50 = 11.45 and 14.53 μg mL−1 , respectively). The active compound was purified using bioassay-guided fractionation and thin layer chromatography, and identified as ursolic acid using high-performance liquid chromatography coupled with a photodiode array and mass spectrometry. The purified compound was strongly inhibitory against the promastigote and amastigote forms of L. infantum and L. major (IC50 = 5.87 and 6.95 μg mL−1 versus 9.56 and 10. 68 μg mL−1 , respectively) without overt cytotoxicity against Raw 264.7 macrophage cells (SI = 13.53 and 11.43, respectively). The commercial compound (ursolic acid) showed similar activity against amastigotes and promastigotes forms of L. infantum and L. major. Moreover, its molecular mode of action against leishmaniasis seems to involve the expression of the ODC and SPS genes involved in thiol pathway. Conclusion: Extracts of M. nervosa can be considered as a potential alternative to antimicrobial and antileishmanial drugs

Identification of Cryptosporidium Species Infecting Humans in Tunisia

International audiencePrevalence and species distribution of Cryptosporidium spp. were determined among 633 immunocompetent children less than five years of age and 75 patients hospitalized for immunodeficiency who lived in northern Tunisia. Microscopy was used for initial screening to detect positive samples and a nested polymerase chain reaction and restriction fragment length polymorphism analysis was used to determine the species. Cryptosporidium spp. was identified in 2.7% of cases (19 stool samples), and there was a significant difference between samples collected from immunocompromised patients and those collected from healthy children (10.7% versus 1.7%). Prevalence was also significantly higher in diarrheal specimens than in formed specimens (6.3% versus 1.6%). Cyptosporidium hominis and C. parvum were responsible for most Cryptosporidium spp. infections (78.9%). Cryptosporidium hominis was more prevalent in children from urban areas than in those from rural areas, and C parvum was found with similar prevalence rates in the two populations. Cryptosporidium meleagridis was identified in four children on farms

Utilization of Grape Seed Flour for Antimicrobial Lipopeptide Production by Bacillus amyloliquefaciens C5 Strain

International audienceAn endophytic Bacillus amyloliquefaciens strain called C5, able to produce biosurfactant lipopeptides with a broad antibacterial activity spectrum, has been isolated from the roots of olive tree. Optimization of antibacterial activity was undertaken using grape seed flour (GSF) substrate at 0.02, 0.2, and 2% (w/v) in M9 medium. Strain C5 exhibited optimal growth and antimicrobial activity (MIC value of 60 μg/ml) when incubated in the presence of 0.2% GSF while lipopeptide production culminated at 2% GSF. Thin layer chromatography analysis of lipopeptide extract revealed the presence of at least three active spots at Rf 0.35, 0.59, and 0.72 at 0.2% GSF. Data were similar to those obtained in LB-rich medium. MALDI-TOF/MS analysis of lipopeptide extract obtained from 0.2% GSF substrate revealed the presence of surfactin and bacillomycin D. These results show that GSF could be used as a low-cost culture medium supplement for optimizing the production of biosurfactants by strain C5

Antioxidant antileishmanial cytotoxic and antimicrobial activities of a local plant Myrtus nivellei from Algeria Sahara

Objective: To study the phytochemical constituents and in vitro biological activities of hydromethanolic extract and fractions from Algerian Sahara Myrtus nivellei (M. nivellei) collected in Hoggar region and to identify the active fraction that can act as an alternative of commonly used antibiotics and as antileishmanial or antioxidant agents. Methods: Phytochemical screening of M. nivellei aerial parts was realised according to the literature. Extract was firstly prepared by using aqueous methanol then fractionated with ethyl acetate and butanol solvents. Total phenolics, tannis and flavonoids, of the hydromethanolic extract and their fractions were determined by Folin–Ciocalteu method as gallic acid equivalents and by aluminium chloride as rutin equivalent respectively. Extract and fractions were tested for their antimicrobial and antiparasital activities against standard bacteria using agar diffusion method and two kinds of leishmania visceral and cutaneous. The antioxidant activities were realized using phosphomolybdenum, FRAP and DPPH tests. Results: Preliminary phytochemical screening exhibited the presence of flavonoids, tannins, saponins, and alkaloids. The experimental results showed that plant extract and fractions were high in phenolic compounds and exhibited an important role as antioxidant, antimicrobial and had a moderate antileishmanial activity. Conclusions: These observations lead us toward more studies in this field, so that we can get more benefits from our local Algerian medicinal plants

Myrtus communis leaf compounds as novel inhibitors of quorum sensing-regulated virulence factors and biofilm formation: In vitro and in silico investigations

Antibiotic resistance of the Gram-negative bacterium Pseudomonas aeruginosa and its ability to form biofilm through the Quorum Sensing (QS) mechanism are important challenges in the control of infections caused by this pathogen. The extract of Myrtus communis (myrtle) showed strong anti-QS effect on Chromobacterium. violaceum 6267 by inhibiting 80 % of the production of violacein pigment at a sub-MIC concentration of 1/8 (31.25 μg/mL). In addition, the extract exhibited an inhibitory effect on virulence factors of P. aeruginosa PAO1 at half MIC (125 μg/mL), significantly reducing the formation of biofilms (72.02 %), the swarming activity (75 %), and the production of protease (61.83 %) and pyocyanin (97 %). The active fraction also downregulated the expression of selected regulatory genes involved in the biofilm formation and QS in the P. aeruginosa PAO1 strain. These genes included the autoinducer synthase genes (lasI and rhlI), the genes involved in the expression of their corresponding receptors (lasR and rhlR), and the pqsA genes. The analysis of the active fraction by HPLC/UV/MS and NMR allowed the identification of three phenolic compounds, 3,5-di-O-galloylquinic acid, myricetin 3-O-α-l-rhamnopyranoside (myricitrin), and myricetin 3-O-(2″-O-galloyl)-ß-d-galactopyranoside. In silico studies showed that 3,5-di-O-galloylquinic acid, with an affinity score of −9.20 kcal/mol, had the highest affinity to the active site of the CviR protein (3QP8), a QS receptor from C. violaceum. Additionally, myricetin 3-O-α-l-rhamnopyranoside (myricitrin) and myricetin 3-O-(2″-O-galloyl)-ß-d-galactopyranoside interact to a lesser extent with 3QP8. In conclusion, this study contributed significantly to the discovery of new QS inhibitors from M. communis leaves against resistant Gram-negative pathogens

Towards the use of Cupressus sempervirens L. organic extracts as a source of antioxidant, antibacterial and antileishmanial biomolecules

Cupressus sempervirens L. is largely used in traditional medicine as an antimicrobial agent. The present study investigated the antioxidant, antibacterial and antileishmanial activities of C. sempervirens organic extracts at different phenological stages. Antioxidant activity was determined by DPPH (2,2-diphenyl-1-picrylhydrazylradical) scavenging assay, ferric reducing power and total antioxidant capacity. The antibacterial activity was evaluated against five clinical strains by disk diffusion method and minimum inhibitory concentration (MIC). The antileishmanial activity was determined against promastigote and amastigote forms of Leishmania (L.) infantum and L. major. Results of antioxidant activity showed that methanolic extract from vegetative stage had the most important activity. The ethyl acetate extract of C. sempervirens from flowering stage was the most active against Bacillus cereus (ATCC 14579) and Staphylococcus aureus (ATCC 25923) with MIC of 100 μg/mL and 50 μg/mL, respectively. Interestingly, this extract exhibited high antileishmanial activity against promastigote form of L. infantum and L. major (IC50 = 1.47 and 2.8 μg/mL, respectively) and amastigote form (IC50 = 3.61 and 5.42 μg/mL, respectively). Furthermore, ethyl acetate extract showed low cytotoxicity on macrophage cells Raw264.7 with selectivity index of 34.15 and 17.93 for L. infantum and L. major, respectively. The identification by HPLC and HPLC-MSn of active extracts of C. sempervirens revealed that major compounds of methanolic extract from vegetative stage and ethyl acetate extract from flowering stage were cupressuflavone and amentoflavone. Based on these results, C. sempervirens extracts could be used as an alternative to chemical drugs for the treatment of oxidative stress and infectious diseases