Antibiotic Medium Research Articles

Heterogeneity in establishment of polyethylene glycol-mediated plasmid transformations for five forest pathogenic Phytophthora species.

Plasmid-mediated DNA transformation is a foundational molecular technique and the basis for most CRISPR-Cas9 gene editing systems. While plasmid transformations are well established for many agricultural Phytophthora pathogens, development of this technique in forest Phytophthoras is lacking. Given our long-term research objective to develop CRISPR-Cas9 gene editing in a forest pathogenic Phytophthora species, we sought to establish the functionality of polyethylene glycol (PEG)-mediated plasmid transformation in five species: P. cactorum, P. cinnamomi, P. cryptogea, P. ramorum, and P. syringae. We used the agricultural pathogen P. sojae, a species for which PEG-mediated transformations are well-established, as a transformation control. Using a protocol previously optimized for P. sojae, we tested transformations in the five forest Phytophthoras with three different plasmids: two developed for CRISPR-Cas9 gene editing and one developed for fluorescent protein tagging. Out of the five species tested, successful transformation, as indicated by stable growth of transformants on a high concentration of antibiotic selective growth medium and diagnostic PCR, was achieved only with P. cactorum and P. ramorum. However, while transformations in P. cactorum were consistent and stable, transformations in P. ramorum were highly variable and yielded transformants with very weak mycelial growth and abnormal morphology. Our results indicate that P. cactorum is the best candidate to move forward with CRISPR-Cas9 protocol development and provide insight for future optimization of plasmid transformations in forest Phytophthoras.

Photodegradation of antibacterial cefotaxime using Mn doped ZnO nanosphere

Elimination of pharmaceutical waste is essential for aquatic system preservation. In this report, the ZnO and Mn-ZnO nanospheres (MZNs) were effectively synthesized via facile co-precipitation method with different Mn doping concentrations as catalyst for photodegradation of cefotaxime. The structure features of the synthesized MZNs were successfully investigated. The obtained data were confirmed the successful fabrication of nanosphere ZnO and MZNs. The designed MZNs with various Mn concentrations were utilized in photodegradation of cefotaxime under optimum conditions. The photodegradation parameters such as antibiotic initial concentrations, catalyst dosage, and medium pH were carried out under UV irradiation. The superior photocatalytic activity was revealed when the MZNs molar ratio is 0.75 mol.%. The results show that the degradation efficiency of cefotaxime was 99% within 2 h at operating condition (initial concentration 30 mg/L, catalyst amount 0.5 g/ L and pH 9). The photodegradation mechanism of antibacterial cefotaxime drug in water is based on superoxide radicals which are considered the main reactive species. The developed MZNs exhibit high stability for removal of cefotaxime that reached to 98% after four times recycling. The MZNs demonstrated a simple and effective model for photocatalytic degradation of pharmaceutical waste.

Improved Antibiotic Activity from Streptomyces monomycini strain RVE129 Using Classical and Statistical Design of Experiments

Many bioactive secondary metabolites with intriguing antibacterial, antiviral, and anticancer properties have been produced by Streptomyces species. The objective of this work is to use conventional and statistical techniques to improve the antibiotic production medium of Streptomyces monomycini RVE129, which was isolated from rhizospheric soil in Hawassa, Ethiopia. The main media components were chosen using the one factor at a time method and the Plackett-Burman design, which was then, further, optimized using the Box-Behnken Design for increased antibiotic production. On ISP4 medium (10 g/L starch, 1 g/L NaCl, 1 g/L MgSO4.7H2O, 2 g/L (NH4) 2SO4, 2 g/L CaCO3and 1 g/L K2HPO4, 0.1 g/L FeSO4·7H2O, 0.1 g/L MnCl2·4H2O, 0.1 g/L ZnSO4·7H2O), S. monomycini RVE129 produced the greatest amount of antibiotics. Starch and soybean meal were found to be the best sources of carbon and nitrogen for the strainRVE129. During the eighth day of incubation under shaking conditions, the best conditions for antibiotic synthesis were determined at a temperature of 30°C and a pH of 7.5. Plackett-Burman design identified K2HPO4, starch, and soybean meal as having the highest influence on antibiotic synthesis with a confidence level above 95%. The yield of producing antibiotics increased by 24.30% when the concentration of critical variables was further improved by using the Box-Behnken Design of the Response Surface approach. The optimum concentration was 20 g/L starch, 7.5 g/L s oybean meal, and 1.25 g/L K2HPO4. To the best of our knowledge, this is the first investigation into medium optimization for the production of the antibiotic from S. monomycini RVE129.

Adherence to Antibiotic Prescription of Dental Patients: The Other Side of the Antimicrobial Resistance.

Since non-adherence to antibiotic therapy can cause several problems, including antimicrobial resistance (AMR) and treatment failures, the present study evaluated adherence to oral antibiotic therapy and AMR awareness among consecutively enrolled dental patients. Data concerning age, gender, socioeconomic status, education level, cohabitation, and general health were retrieved from medical records. AMR awareness was investigated through direct questions and adherence to antibiotic treatment was assessed through a modified Italian version of the Morisky medical scale-8 items. Participants’ characteristics were analyzed in relation to treatment adherence and AMR, using a Χ2 independence test (significance level of α <0.1). Dental patients generally showed a low (51.82%) adherence to oral antibiotic therapy, and medium and high adherence was reported only by 29.37% and 18.81% of participants. Treatment adherence was similar in relation to participants’ gender and age but significantly lower in subjects with only secondary school graduation and higher in participants with higher education levels. Non-cohabitants were significantly more adherent than cohabitants. AMR awareness was declared by 42.15% of males and 38.70% of females: 56.52% of dental patients aware of AMR were 18–38 years old, 35.20% were 39–59 years old, and 26.95% were aged between 60 and 80. Further studies are needed to develop adequate strategies, expanding dental patients’ knowledge of AMR, thus optimizing the benefits and reducing the risks of antibiotic administration in dental patients.

The Impact of Pre- and Probiotic Product Combinations on Ex vivo Growth of Avian Pathogenic Escherichia coli and Salmonella Enteritidis

Due to the global spread of antibiotic resistance, there is a strong demand to replace antimicrobial growth promotors in livestock. To identify suitable additives that inhibit the growth of avian pathogenic Escherichia coli O1/O18 and Salmonella enterica serotype Enteritidis strains, an ex vivo screening was performed. Inulin and fructooligosaccharides (FOS) were investigated as prebiotics. Enterococcus faecium and Bacillus coagulans served as probiotic strains. Firstly, the pathogen was anaerobically incubated in caecal digesta from different broiler breeder flocks with the addition of feed additives. Secondly, subsamples of these suspensions were incubated in an antibiotic medium for selective growth of the pathogen. During this step, turbidity was recorded, and lag times were calculated for each pathogen as readout of growth inhibition. Combinations of E. faecium with inulin or FOS significantly extended the lag time for E. coli compared to control. Moreover, older age was a significant factor to enhance this inhibitory effect. In contrast, the combination of FOS and B. coagulans showed shorter lag times for S. Enteritidis. Our results indicate that the E. faecium strain with prebiotics may inhibit the pathogen proliferation in the studied poultry flocks. Furthermore, our results suggest that prophylactic treatments should be assigned by feed additive, age and animal origin.

Bicarbonate modulates delafloxacin activity against MDR Staphylococcus aureus and Pseudomonas aeruginosa.

To investigate the utility of recently approved delafloxacin and other fluoroquinolones against leading MDR bacterial pathogens under physiologically relevant conditions. MIC and MBC assays were conducted for MDR strains of Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae in the standard antibiotic susceptibility testing medium CAMHB, amended Roswell-Park Memorial Institute tissue culture medium (RPMI+) or 20% fresh human whole blood. In vivo correlation of in vitro findings was performed in a murine P. aeruginosa pneumonia model. Mechanistic bases for the findings were explored by altering media conditions and with established fluoroquinolone accumulation assays. Fluoroquinolone MICs were increased in RPMI+ compared with CAMHB for all four MDR pathogens. Specifically, delafloxacin MICs were increased 32-fold versus MDR S. aureus and 8-fold versus MDR P. aeruginosa. MBC assays in 20% human whole blood and a murine MDR P. aeruginosa pneumonia model both confirmed that delafloxacin activity was reduced under physiological conditions. Bicarbonate (HCO3-), a key component of host physiology found in RPMI+ but absent from CAMHB, dictated delafloxacin susceptibility in CAMHB and RPMI+ by impairing its intracellular accumulation. Standard in vitro antibiotic susceptibility testing conditions overpredicted the effectiveness of delafloxacin against MDR pathogens by failing to capture the role of the biological buffer HCO3- to impair delafloxacin accumulation. This work showcases limitations of our current antibiotic susceptibility testing paradigm and highlights the importance of understanding host microenvironmental conditions that impact true clinical efficacy.

Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228

BackgroundPenicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus.ResultsPenicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ∆cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome.ConclusionsIn this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods.

Residues of streptomycin antibiotic in commercial layers in Abeokuta and Ibadan metropolis

Old layers sold for human consumption in six open markets in Abeokuta and Ibadan metropolis of Ogun and Oyo States of Nigeria were analyzed for the presence of residues of streptomycin antibiotic. One hundred and twenty samples comprising of muscle (thigh and breast), liver and kidney tissues were subjected to microbiological assay technique for the detection of residues of the antibiotic. The assay was done on antibiotic medium 5 agar with Bacillus subtilis (ATCC 6633) organism. Results showed that 34.17% of the sampled birds were positive for the presence of streptomycin antibiotic. The concentration of the residues ranged between 0.18µg/g and 0.91µg/g. The mean residue levels recorded among the organs are not significantly different (P&gt;0.05). Some of the muscle samples recorded residue concentration levels higher than the W.H.O recommended maximum residue level for streptomycin antibiotic in muscle tissue of food animals. In view of the importance of antibiotics in the treatment of bacterial diseases in both human and animals, it is advocated that the use of these drugs in food animals be done judiciously.

Development and Validation of a Microbiological Agar Assay for Determination of Thiamphenicol in Soft Capsules

Background: Thiamphenicol belongs to the amphenicol class of antibiotic and possesses a broad-spectrum antimicrobial activity. An alternative microbiological assay for quantification of thiamphenicol in pharmaceutical formulations has not yet been reported in the literature. Objective: This study aimed to develop and validate an agar diffusion method for quantification of thiamphenicol in soft capsules. Methods: The assay was based on the inhibitory effect of thiamphenicol on the following: a strain of Kocuria rhizophila ATCC 9341, used as the test microorganism, Antibiotic 1culture medium, phosphate buffer pH 6, 0, inoculum at a concentration of 1%, as well as standard and sample solutions at the concentrations of 20.0, 40.0 and 80.0 μg mL-1. Results: The method validation yielded good results for the parameters of linearity, precision, accuracy, robustness and selectivity. The experimental statistic results were analyzed using analysis of variance (ANOVA). The method was found to be linear (r2 = 0.9992) in the range of 20-80 μg mL-1, precise (inter-assay R.S.D = 0.09%), accurate (R.S.D. = 4.65%), specific, and robust. Conclusion: The results demonstrated the validity of the proposed bioassay, which allows for reliable quantification of thiamphenicol in a pharmaceutical sample. An alternative methodology for thiamphenicol determination in routine quality control has been reported herein.

Achromobacter sp. FB-14 harboring ACC deaminase activity augmented rice growth by upregulating the expression of stress-responsive CIPK genes under salinity stress.

Soil salinity is one of the major plant growth and yield-limiting constraints in arid and semi-arid regions of the world. In addition to the oxidative damage, increasing salt stress is associated with elevated cellular ethylene levels due to the synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) in large amounts. The objective of the current study was to elucidate the inoculation effect of an ACC deaminase (ACCD)-producing phytobeneficial strain Achromobacter sp. FB-14 on rice plants to alleviate the salinity effects by upregulation of the stress-responsive CIPK genes. The strain FB-14 was isolated by using nutrient agar medium at 855mM NaCl concentration and it was taxonomically identified as Achromobacter sp. with more than 99% 16S rRNA gene sequence similarity with many Achromobacter species. The strain FB-14 demonstrated substantial in vitro potential for ACCD activity, synthesis of indole compounds, and phosphate solubilization up to 100mM NaCl concentration in the culture medium. The gene corresponding to ACCD activity (acdS) was amplified and sequenced in order to confirm the inherent enzyme activity of the strain at a molecular level. The rifampicin-resistant derivative of strain FB-14 was recovered from the rice rhizosphere on antibiotic medium up to 21days of sowing. Moreover, the strain FB-14 was inoculated on rice plants under salinity and it not only enhanced the growth of rice plants in terms of root and shoot length, and fresh and dry weight, but also upregulated the expression of stress-responsive CIPK genes (OsCIPK03, OsCIPK12, and OsCIPK15) according to the results of qRT-PCR analysis. To the best of our knowledge, this is the first report deciphering the role of plant-beneficial Achromobacter strain relieving the rice plants from salt stress by promoting the growth and enhancing the expression of stress-responsive CIPK genes.

Tenebrio molitor-mediated entomopathogenic fungal library construction for pest management

Entomopathogenic fungi are soil-dwelling microorganisms that infect host insects, and some have been used as biological control agents. Construction of an entomopathogenic fungal library provides a strong platform for the development of highly effective biopesticides, but the traditional method using antibiotic media has low isolation efficiency. Herein, to increase the efficiency of isolation, a Tenebrio molitor pathogenicity-based fungal collection method was used for the construction of a library. The isolation efficiency using the T. molitor larvae-based pathogenicity assay was 55.4%, which was significantly higher than the method of antibiotic medium with dodine (6.0%). We named it the Jeonbuk (Chonbuk) National University Entomopathogenic Fungi (JEF) library. It consisted of 279 isolates belonging to 12 genera and 29 species. Main features of the species-representative 15 isolates were characterized in terms of morphology, virulence, conidiogenesis, thermotolerance and production of biologically active materials such as virulence-related enzymes. Some of the species-representative isolates showed higher virulence against T. molitor, Riptortus pedestris and Plutella xylostella in laboratory conditions. The production of enzymes such as chitinase, Pr1 protease and lipase all related to pathogenesis in LB medium was higher than in the SDB and PDB media. In the thermotolerance assay, a Purpureocillium lilacinum isolate showed higher thermotolerance than other isolates. This work reports an efficient library method using T. molitor for building a library for development of fungal biopesticides for pest management.

Removal of Amoxicillin and Sulfanomide by Freshwater Bacteria in Sri Lanka

Antibiotics are an important group of pharmaceuticals used in human and animal health care. Most of the antibiotics are prone to release to the environment due to improper usage. This has resulted in bacterial resistance development and toxicity in aquatic communities. Present study reports the biodegradation of amoxicillin (AMX) and sulfanomide (SUF) by Bacillus cereus, Enterobacter ludwigii and Enterobacter sp. strains which were previously reported as crude oil degraders. Different concentrations of AMX and SUF (0, 60, 120, 180, 240, 300, 360, 420 ppm) were used to detect minimum inhibition concentration (MIC) by standard pour plate method. AMX and SUF degradation kinetics were studied by introducing 0.5 ml of overnight starved bacterial suspensions into sterile antibiotic medium in triplicate at final concentration of 60 and 120 ppm respectively. The samples were incubated at 280 C shaking at 100 rpm and 0.5 ml sub-sample aliquots were removed at two days interval for a period of 14 days. Analyses of antibiotics were performed by high performance liquid chromatography (HPLC). The MIC values for SUF and AMX were detected as 240, 420 ppm for B. cereus, 120, 360 ppm for E. ludwigii and 180 and 300 ppm for Enterococcus sp. respectively. After 14 days of incubation complete removal of AMX and 80% degradation of SUF was recorded by bacterium B. cereus. E. ludwigii showed 75% degradation of AMX and 60% degradation of SUF where Enterobacter sp. showed degradation of both AMX (80%) and SUF (70%) respectively. Thus, the present study illustrate antibiotics degradation potential of microbial community is important to understand their role in removal of antibiotics from the natural environment. Keywords: Biodegradation, Minimum Inhibitory Concentrations (MICs), Amoxicillin, Sulfanomide, Bacillus cereus, Enterobacter ludwigii, Enterobacter sp.

Effect of carbon on whole-biofilm metabolic response to high doses of streptomycin.

Biofilms typically exist as complex communities comprising multiple species with the ability to adapt to a variety of harsh conditions. In clinical settings, antibiotic treatments based on planktonic susceptibility tests are often ineffective against biofilm infections. Using a CO2 evolution measurement system we delineated the real-time metabolic response in continuous flow biofilms to streptomycin doses much greater than their planktonic susceptibilities. Stable biofilms from a multispecies culture (containing mainly Pseudomonas aeruginosa and Stenotrophomonas maltophilia), Gram-negative environmental isolates, and biofilms formed by pure culture P. aeruginosa strains PAO1 and PAO1 ΔMexXY (minimum planktonic inhibitory concentrations between 1.5 and 3.5 mg/l), were exposed in separate experiments to 4000 mg/l streptomycin for 4 h after which growth medium resumed. In complex medium, early steady state multispecies biofilms were susceptible to streptomycin exposure, inferred by a cessation of CO2 production. However, multispecies biofilms survived high dose exposures when there was extra carbon in the antibiotic medium, or when they were grown in defined citrate medium. The environmental isolates and PAO1 biofilms showed similar metabolic profiles in response to streptomycin; ceasing CO2 production after initial exposure, with CO2 levels dropping toward baseline levels prior to recovery back to steady state levels, while subsequent antibiotic exposure elicited increased CO2 output. Monitoring biofilm metabolic response in real-time allowed exploration of conditions resulting in vulnerability after antibiotic exposure compared to the resistance displayed following subsequent exposures.

Antibiotics supplemented culture media can eliminate non-specific bacteria from human semen during sperm preparation for intra uterine insemination.

RATIONALE:Bacterial flora can be isolated from many semen samples of subfertile males. Bacteriospermia can compromise the outcome of intra uterine insemination (IUI) by contaminating the post-processed sperm sample.OBJECTIVES:The objective of the present study is to determine the efficacy of penicillin and streptomycin in eliminating the bacteria from semen samples in the sperm processing procedure, and to assess the effects of antibiotics on sperm motility, survivability, and pregnancy rates.DESIGN AND SETTINGS:A prospectively controlled study was carried out using couples undergoing IUI with their informed consent.INTERVENTION:Sperm processing using the swim-up technique in penicillin and streptomycin supplemented culture medium.SUBJECTS AND METHODS:Couples were consecutively allocated in two groups for sperm processing (a) Group AB+ (antibiotics supplemented culture medium, n = 33) and (b) Group AB− (antibiotic free culture medium, n = 33). Semen culture was performed before and after sperm processing. Sperm motility was assessed immediately after processing and after 24 h of incubation.RESULTS:Bacterial isolates were found in 20 (60.6%) and 22 (66.1%) of samples before processing in Groups AB+ and AB− respectively. Addition of antibiotics resulted in completely eliminating non-specific bacteria from semen samples without affecting sperm motility. In vitro survival rate of sperm enhanced in AB+ group compared with AB− group (motile sperm after 24 h), 62.21% (standard deviation [SD]: 37.27) versus 41.36% (SD: 30.78), P = 0.012. Pregnancy rate, was comparable between two groups (9% in Group AB+ vs. 6% in Group AB−, P = 0.45).CONCLUSION:Penicillin streptomycin combination could completely eliminate non-specific bacteria from semen samples during sperm processing in this population. The types of antibiotics and dosage used did not seem to have any harmful effects on human sperm.

Development and Validation of Microbiological Assay for Ceftriaxone and its Application in Photo-stability Study

The aim of this work is to develop and validate a microbiological assay for ceftriaxone with a view to the employment of this method to assess photo-stability in commercial products. The experimental conditions included: (1) ceftriaxone standard solution in concentrations of 2.6 μg/mL, 3.2 μg/mL, 4.0 μg/mL, 5.0 μg/mL and 6.3 μg/mL, using phosphate buffer pH 6.0 as diluent, (2) Petri dishes prepared by adding 21-mL of antibiotic medium as base layer and 4-mL of the same medium, inoculated with Bacillus subtilis (ATCC 6633) in a proportion of 0.5% and (3) incubation at 37ºC for 18 hours. The method showed specificity, good linearity in the range from 2.6 to 6.3 μg/mL, as well as good precision (RSD repeatability of 1.7% and RSD intermediate precision of 2.2%) and accuracy (recovery of 101.1%). Commercial samples of ceftriaxone were exposed to light in their primary and secondary packing, being afterwards directly exposed to light. The potencies of commercial samples were then determined by the developed and validated microbiological assay. The results indicate that primary and secondary packing provide satisfactory protection of ceftriaxone from light. Keywords: Agar diffusion, Antibiotic assay, Ceftriaxone, Cephalosporin, Dosage form, Method development, Method validation, Microbial assay, Photo-stability, Stability study

Screening for actinomyces isolated from soil with the ability to inhibit Xanthomonas oryzae pv. oryzae causing rice bacterial blight disease in Vietnam

Bacterial blight disease caused by Xanthomonas oryzae pv. oryzae ( Xoo ) is one of the major diseases in rice culture of Northen Vietnam, as well as other rice-growing regions of the world. In this study, we isolated and screened for actinomycete strains from Vietnam with the ability to inhibit Xoo isolates from northern Vietnam. From 90 actinomycete strains taken from soil in northern Vietnam in 2010, we screened for their antagonistic activity against 10 races of Xoo causing rice bacterial blight disease. Three actinomycete strains were found to inhibit all 10 Xoo races. Among the three strains, a strain namely VN10-A-44 was shown not to have the ability to produce toxic compounds and was selected for further study. The strain was identified as Streptomyces virginiae by 16S ribosomal RNA gene sequencing. We replaced soybean meal with tofu waste in antibiotic producing medium to improve antagonistic activity of VN10-A-44 against the Xoo pathogen and to make use of tofu waste for large-scale fermentation of VN10-A-44. We found that replacing soybean meal with 20 and 30 g of tofu waste/litter in the antibiotic producing medium gave the largest inhibition zone against the Xoo pathogen. Key words : Xanthomonas oryzae pv. oryzae , rice bacterial blight disease, Streptomyces virginiae , Vietnam.

Integument Mycobiota of Wild European Hedgehogs (Erinaceus europaeus) from Catalonia, Spain

There are some reports about the risk of manipulating wild hedgehogs since they can be reservoirs of potential zoonotic agents like dermatophytes. The aim of this study was to describe the integument mycobiota, with special attention to dermatophytes of wild European hedgehogs. Samples from spines and fur were cultured separately in Sabouraud dextrose agar (SDA) with antibiotic and dermatophyte test medium (DTM) plates. Nineteen different fungal genera were isolated from 91 cultures of 102 hedgehogs. The most prevalent genera were Cladosporium (79.1%), Penicillium (74.7%), Alternaria (64.8%), and Rhizopus (63.7%). A lower prevalence of Aspergillus (P = 0,035; χ 2 = 8,633) and Arthrinium (P = 0,043; χ 2 = 8,173) was isolated during the spring time and higher frequencies of Fusarium (P = 0,015; χ 2 = 10,533) during the autumn. The prevalence of Acremonium was significantly higher in young animals (70%, 26/37) than in adults (30%, 11/37) (P = 0,019; χ 2 = 5,915). Moreover, the majority of the saprophytic species that grew at the SDA culture were also detected at the DTM. Finally, no cases of ringworm were diagnosed and no dermatophytes spp. were isolated. Concluding, this study provides the first description of fungal mycobiota of the integument of wild European hedgehogs in Spain, showing a large number of saprophytic species and the absence of dermatophytes.

Enquête sur les conditions analytiques d’un examen de mycologie médicale auprès des membres de la Société française de mycologie médicale

The objective of the survey was to describe the practices of clinical laboratories in terms of cultures in medical mycology. We have implemented this project within the members of the French Society for Medical Mycology (SFMM) to evaluate the analytical processes of the mycological examination in our laboratories. This preliminary study would help to suggest the future French guidelines.A questionnaire regarding the processing of mycology analysis was sent to the 227 members of the SFMM in 2009. The data involved 21 types of samples, direct microscopic examination with or without colouring and the reagents, the number of culture media, the types of media (Sabouraud, Sabouraud antibiotic, Sabouraud cycloheximide and chromogenic medium), temperature and duration of the incubation (days) and the existence of a first result before the end of the incubation period. The analytical processes were compared to an accredited laboratory according to EN ISO 15189.A great heterogeneity was observed in the 36 forms from 27 (75%) laboratories belonging to university hospitals among the 38 existing in France. As for deep samples, two microscopic exams were performed, only one was usually done. A more sensitive technique was preferred to the wet-mount for some samples. Routine samples are often inoculated on a chromogenic media. For deep samples two medium are inoculated (chromogenic media, Sabouraud and antibiotics). If the temperature of incubation is unique, 30°C was chosen. A temperature of 37°C was preferred for samples where Candida spp. is selected. When there are two temperatures of incubation, 27°C and 37°C were preferred.Each biologist can compare his proceedings to the other laboratories and to a laboratory already accredited. The question is to find the best strategies for each medical mycology specimen. They will aid the process of accreditation according to EN ISO 15189, which now applies in all laboratories in Europe.

Detection of Leptospira interrogans Serovar Grippotyphosa in Bovine Embryos In Vitro Fecundated after Pathogens Transmission Control Treatment.

The advances in biotechnology in animal reproduction are intended to maximize reproductive efficiency in the distribution of genetic material of high value. However, these techniques should also be used as tools to control infectious diseases transmitted by agents possibly associated with gametes and embryos. Thus, treatment of embryos with trypsin or antibiotics in alternating washes with culture medium, established by the International Society of Embryo Transfer (IETS), aims to remove and/or inactivate infectious agents that may interfere in the final product. The aim of this study was to detect, by dark field optical microscopy, the presence of Leptospira interrogans serovar Grippotyphosa in bovine embryos in vitro fecundated experimentally exposed to pathogen during the maturation period, after the standardized treatment by IETS. Oocytes were aspirated from ovaries of slaughtered cows and ones with the intact zona pellucida were selected and matured. After 20 hours, the oocytes were divided into two groups: the control (n=360) and exposed to 30 µL of the pathogen at 6,3 x 105/µL (n=360). The semen was treated by the technique of discontinuous Percoll gradient, and the sperm concentration was adjusted to approximately 100 thousand sperm for each oocyte. After the period of fecundation, the embryos were subjected to the treatments with antibiotic, trypsin and TCM199 medium. The results showed absence of the pathogen in all embryos after washes. Thus, we conclude that the treatment suggested by the IETS used in bovine embryos infected with L. interrogans serovar Grippotyphosa was 100% effective. However, more studies of sanitary control in bovine embryos in vitro production (IVP) should be performed, since the action of the treatments may vary according to species, virulence and pathogen concentration. This research was supported by CNPq and Vitrocell/Embriolife. (poster)

In Vitro Activity of Amphotericin B Against Candida lusitaniae Clinical Isolates

" In Vitro Activity of Amphotericin B Against Candida lusitaniae Clinical Isolates." Journal of Chemotherapy, 22(1), pp. 71–72