Isolation of Candida spp. from mastitic bovine milk in Brazil

The purpose of this study was to isolate yeast (Candida) from the quarter milk of cow udders from 37 dairy farms in Brazil and to identify the different species involved in mastitis. The samples were collected between October 2002 and February 2003. Two-hundred-and-sixty milk samples from cows with clinical and subclinical mastitis were examined. Milk samples were plated onto Blood agar, Mac Conkey agar and Sabouraud dextrose agar. Forty-five (17.3%) samples were positive for the genus Candida. The Candida species isolated were C. krusei (44.5%), C. rugosa (24.5%), C. albicans (8.9%), C. guilliermondii (8.9%), and others (13.2%). We also isolated Escherichia coli (26.5%), coagulase-positive Staphylococcus (25.0%), Streptococcus spp. (8.1%), Enterobacter spp. (8.1%), and other fungi (8.1%), among others.

Semi-selective culture medium for Curtobacterium flaccumfaciens pv. flaccumfaciens isolation from bean seeds

Curtobacterium wilt has become an important disease of beans in several localities in the country. Its causal agent, Curtobacterium flaccumfacciens pv. flaccumfaciens (Cff), survives and is disseminated through seeds. To date, few studies have been conducted with the objective of developing an effective and low-cost culture medium to isolate this bacterium from bean seeds, for health analysis purposes. Usually, the culture media employed for coryneform bacteria isolation contain specific carbon sources and antimicrobial products not available in the Brazilian market. A culture medium known as MSCFF was developed (peptone - 5 g, meat extract - 3 g, sucrose - 5 g, agar 15 g, skim milk powder* - 5 g. Congo red* - 0.05 g-, chlorothalonil* - 0.01 g, thiophanate methyl* - 0.01 g, nalidixic acid* - 0.01 g, nitrofurantoin* - 0.01 g. oxacillin* 0.001 g, sodium azide* - 0.001 g and distilled water q.s. 1L; *added after autoclaving the basal medium), which has the ability to inhibit growth of a large amount of saprophytic bacteria, but with low supressivity to Cff isolates. The MSCFF medium was highly effective for Cff isolation from naturally infected bean seeds and could be used for routine detection of this bacterium in bean seeds.

Evaluation of the in vitro activity of cefepime compared to other broad-spectrum cephalosporins against clinical isolates from eighteen Brazilian hospitals by using the Etest

The in vitro activity of cefepime was compared to that of ceftazidime, ceftriaxone, and cefotaxime in a multicenter study involving 10 clinical microbiology laboratories and clinical isolates from 18 Brazilian hospitals from 7 cities (4 states). A total of 982 isolates consecutively collected between December 1995 and March 1996 were susceptibility tested by using Etest and following the NCCLS procedures for agar diffusion tests. The cefepime spectrum was broader than that of the other broad-spectrum cephalosporins against both Gram-negative rods and Gram-positive cocci. Cefepime tons particularly move active against Enterobacter sp. (MIC90, 2 mu g/ml), Serratia sp. (MIC90, 2 mu g/ml) and oxacillin-susceptible Staphylococcus aureus (MIC90, 3 mu g/ml). Against Pseudomonas aeruginosa, cefepime (MIC90 16 mu g/ml) was slightly more active than ceftazidime (MIC90 32 mu g/ml) and 8- to 16-fold more active than ceftriaxone or cefotaxime (MIC90 >256 mu g/ml). Our results show that nosocomial bacteria, especially Gram-negative rods, have a high rate of cephalosporin resistance in Brazil. However, part of these resistant bacteria remains susceptible to cefepime. The Etest was shown to be an excellent method for multicenter studies of the in vitro evaluation of new antimicrobial agents. (C) 1997 Elsevier B.V.

Microbiological analyses of poultry litter used for ruminant feeding

The aims of this study were to estimate the changes in total bacterial counts (TBC) in poultry litter samples, consisting of rice hulls, after storage, and to identify pathogenic bacteria. For the countings Plate Count agar (Difco) was used. Enrichment and selective media such as blood agar, MacConkey, Baird Parker, brain and heart agar, and egg yolk solid media, and cooked meat and brain and heart infusion, incubated under aerobic or anaerobic conditions were used to isolate Staphylococcus aureus, Salmonella sp, Clostridium perfringens, C. botulinum, C. chauvoei, Campylobacter sp, Escherichia coli, and Corynebacterium sp. Litter samples were collected from the houses of the Veterinary School experimental aviary. A fully randomized experimental design was used with four treatments and four replications, for a total of 16 samples. A decrease in TBC was detected when treatment T1 (zero days of storage) was compared with treatments T2 (14 days of storage). on the other hand the treatments T3 (28 days of storage) and T4 (42 days of storage) presented significantly superior counting in relation to treatment T1. Some pathogenic bacteria of Enterobacteriaceae such as Escherichia coil, Proteus, Arizona, Providencia, Edwardsiella, as well as Staphylococcus aureus, S. epidermidis, different species of genus Clostridium as C. perfringens, C. sordelli, C. chauvoei, C. tetani and C. novyi as well as some strains of Corynebacterium pyogenes were isolated.

Morphological and molecular characterization of Colletotrichum spp. from citrus orchards affected by postbloom fruit drop in Brazil

Brazilian isolates of Colletotrichum spp. from citrus orchards affected by postbloom fruit drop were examined for colony colour, mycelial growth, benomyl-resistance, pathogenicity, and genetic variability by random amplified polymorphic DNA (RAPD) analysis. All isolates were obtained from flowers and persistent calyxes from different citrus hosts from São Paulo, Brazil. DNA polymorphisms detected after amplification with random 10-mer primers were used to classify the isolates into two groups. Group I isolates grew rapidly on potato-dextrose agar (PDA) and were sensitive to benomyl, and group II isolates grew slowly on PDA and were benomyl-resistant. Colletotrichum acutatum was analyzed by RAPD and had high genetic similarity with group II isolates of Colletotrichum from citrus. Probably, the group I is C, gloeosporioides and group II is C. acutatum.

Evaluation in vitro of the antagonistic substances produced by Lactobacillus spp. isolated from chickens

To determine the inhibitory capacity of lactic acid bacteria due to the action of antagonistic substances, we tested 474 isolates of Lactobacillus from the crop and cecum of chickens against gram-positive and gram-negative indicator microorganisms by the spot-on-the-lawn and well-diffusion antagonism methods. of the 474 isolates, 265 demonstrated antimicrobial activity against the indicator microorganisms. Isolates identified as L. reuteri, L. salivarius, or Lactobacillus spp. inhibited Enterococcus faecalis, E. faecium, Listeria monocytogenes, and Salmonella spp. but not L. casei, L. delbrueckii, L. fermentum, or L. helveticus by the well-diffusion simultaneous antagonism method under anaerobic incubation conditions. The antagonistic substances produced by some of the Lactobacillus isolates were inactivated after treatment by proteolytic enzymes, which suggested that the substances could be antimicrobial peptides or bacteriocins.

Atividade antimicrobiana de Lactobacillus acidophilus, contra microrganismos patogênicos veiculados por alimentos

The antimicrobial activity of a commercial probiotic culture, Lactobacillus acidophilus (La5), was tested against two foodborne pathogens, Escherichia coli and Staphylococcus aureus. The antagonistic effect of the probiotic culture in vitro was performed by applying both Multilayer Agar Plate and Agar Well Diffusion methods. The results indicated that the inhibitory substance present on 72 hours culture broth supernatant was extracellular and diffusible. The incubation period of the lactic acid bacteria on MRS Broth, at 3 7 degrees C in aerobic conditions, for the highest lactic acid production (1,08 g/%) was 72 hours, which gave a minimum pH value of the supernatant (3,90) and the best inhibition results by the Well Diffusion Agar Assay, showing inhibition zone diameters of 14,75mm and 15,0mm for E. coli and S. aureus, respectively. The inhibitor compound was not sensitive to proteolytic enzyme and freezing, but was totally inactivated when the supernatant was neutralized with NaOH 1 N solution. The results suggest that the inhibitory activity was due to the lactic acid concentration and the low pH of the probiotic culture broth.

A COMPARATIVE-STUDY OF 4 DIFFERENT STAINING METHODS FOR ESTIMATION OF LIVE YEAST FORM CELLS OF PARACOCCIDIOIDES-BRASILIENSIS

A comparative study of four different staining methods for estimation of live yeast form cells of Paracoccidioides brasiliensis was carried out. The staining methods used were fluorescent staining, vital dye exclusion tests with erythrosin B and by Janus green and lactophenol cotton blue staining. Colony forming units (cfu) of the yeast form of eight P. brasiliensis isolates on brain heart infusion agar (BHIA) supplemented with 4% horse serum plus 5% P. brasiliensis cell extract (BHIA + HS + EXT) were examined for reliability of staining in determining the number of live fungal units in eight different isolates. Cfu on BHIA + HS + EXT plates showed an excellent plating efficiency over 96% in all isolates tested. The percentage of the live cells indicated by fluorescent staining (FL) or vital dye exclusion test with erythrosin B (EB) or Janus green (JG-1) was lower than that of cfu. By contrast, the percentage due to modified dye exclusion test with Janus green (JG-2) and that due to lactophenol cotton blue staining (LPCB) showed a close correration to that of cfu. Our results indicate that the modified dye exclusion test with Janus green and lactophenol cotton blue staining are useful for estimating cell viability of yeast form cells of P. brasiliensis.

Cerebriform colonies of Paracoccidioides brasiliensis isolated from nine-banded armadillos (Dasypus novemcinctus) at room temperature

Twelve isolates of Paracoccidioides brasiliensis generated cerebriform colonies at room temperature on potato glucose agar slants (PDA). These isolates contained abundant chlamydospores and yeast-like cells and are a subset of the 65 isolates obtained from nine-banded armadillos (Dasypus novemcinctus). They grew as a yeast form with typical multiple buddings at 37 degreesC on brain heart infusion agar supplemented with 1% glucose. After replating on PDA and culturing at room temperature for 2 months, the mutants appeared as cottonous colonies, which indicated that the morphological characteristics were unstable.

Identification and characterization of Colletotrichum spp. affecting fruit after harvest in Brazil

Colletotrichum spp. cause anthracnose in various fruits post-harvest and are a particularly important problem in tropical and subtropical fruits. The disease in fruits of avocado, guava, papaya, mango and passion fruit has been reported to be caused by C. gloeosporioides, and in banana by C. musae. In subtropical and temperate crops such apple, grape, peach and kiwi, the disease is caused by C. acutatum. The variation in pathogenic, morphological, cultural and molecular characteristics of Brazilian isolates of Colletotrichum acutatum Simmonds and isolates from post-harvest decays of avocado, banana, guava, papaya, mango and passion fruit was evaluated. The fruits were inoculated with mycelium of C. acutatum, Colletotrichum spp. and C. musae on a disc of potato dextrose agar. The morphological, cultural and molecular characteristics studied were conidia morphology, colony growth at different temperatures, colony coloration and PCR with primers CaInt2 and ITS4 for C. acutatum and CgInt and ITS4 for C. gloeosporioides. C. acutatum was pathogenic to avocado, guava, papaya, mango and passion fruit, but it was not pathogenic to banana. The morphological, cultural and molecular studies indicated that the avocado, papaya, mango and passion fruit isolates were C. gloeosporioides. The natural guava isolate was identified as C. acutatum, which had not been found previously to produce anthracnose symptoms on guava in Brazil.

Prevalence of enzootic bovine leukosis among buffaloes (Bubalus bubalis) in the Ribeira valley region state of São Paulo, Brazil

The prevalence of enzootic bovine leukosis (EEL) among female buffaloes reared in 15 herds of the Ribeira Valley, Brazil, was zero. Serum samples(470)were submitted to agar gel immunodiffusion (AGID) with a glycoprotein antigen(gp51) for the diagnosis of EBL. The fact that no seroreactive animals were detected may probably be due to the predominantly extensive management of buffaloes and their little or no contact with cattle, the major source of EEL infection.

An experimental inoculation system to study citrus-Xylella fastidiosa interactions

Difficulties in reproducing the citrus variegated chlorosis (CVC) disease symptoms in expertmental plants have delayed implementation of studies to better understand the essential aspects of this important disease. In an extensive Study, cultivars of sweet orange (Citrus sinensis) were inoculated with Xylella fastidiosa using procedures that included root immersion, and stein absorption, pricking, or infiltration of the inoculum into plants of different ages. Inoculum consisted of 5-day-old cultures or cell suspensions of CVC strain 9a5c diluted in phosphate-buffered saline. Inoculated plants and controls were grown, or transferred just after inoculation, to 5-liter pots or 72-cell foam trays. Approximately 4, 5, 9, and 12 months after inoculation, leaves were collected and processed for polymerase chain reaction analysis or X. fastidiosa isolation on BCYE agar medium. Root immersion and stem inoculation of 4- and 6-month-old plants resulted in low percentages of symptomatic (0 to 7%) and plants positive by isolation (0 to 9%). Pinpricked or injected stems of I-month-old seedlings resulted in high percentages of plants symptomatic (29 and 90% in Pera Rio, 75, 59, and 83% in Valencia, and 77% in Natal) or positive by isolation (26 and 93% in Pera Rio, 98, 96, and 83% in Valencia, and 77% in Natal), In foam trays, the seedlings grew less, the incubation period was shorter. and disease severity was higher than in pots. This system allows testing of higher numbers of plants in a reduced space with a more precise reproduction of the experimental conditions.

Paracoccidioides brasiliensis (Lutz, 1908) isolado por meio de hemocultura em um paciente portadora de símdrome de imunodeficiência adquirida (SIDA)

A case of an AIDS-patient with positive blood culture for Paracoccidioides brasiliensis is reported. The patient was a 29 years old male born in Nova Londrina (Parana State, Brazil) who presented splenomegaly and fever of unknown origin. Three blood cultures were performed, each one of them for aerobic bacteria, mycobacteria and fungi. Cultures for aerobic bacteria and mycobacteria were negative. However, the yeast phase P. brasiliensis was isolated from two cultures in BHI agar, 20 days after inoculation in Negroni medium. The patient was classified in group V according to the Centers for Disease Control (CDC) criteria for AIDS, due to a Pneumocystis carinii pneumonia. Treatment was discontinued due to an individual decision of the patient on the second dose of amphotericin B. This antibiotic was replaced by ketoconazole in the daily dose of 800 mg. The patient died one year after the isolation of P. brasiliensis on blood culture.

Photosensitization of different Candida species by low power laser light

The aim of this study was to evaluate the effects of the laser radiation (685 nm) associated with photosensitizers on viability of different species of Candida genus. Suspensions of Candida albicans, Candida dubliniensis, Candida krusei and Candida tropicalis, containing 106 viable cells per milliliter were obtained with the aid of a Neubauer's chamber. From each species, 10 samples of the cell suspension were irradiated with diode laser (685 nm) with 28 J/cm(2) in the presence of methylene blue (0.1 mg/ml), 10 samples were only treated with methylene blue, 10 samples were irradiated with laser in the absence of the dye, 10 samples were treated with the dye and irradiated with laser light and 10 samples were exposed to neither the laser light nor to the methylene blue dye. From each sample, serial dilutions of 10(-2) and 10(-3) were obtained and aliquots of 0.1 ml of each dilution were plated in duplicate on Sabouraud dextrose agar. After incubation at 37 degrees C for 48 h, the number of colony-forming units (CFU/ml) was obtained and data were submitted to ANOVA and Tukey's test (p < 0.05). Laser radiation in the presence of methylene blue reduced the number of CFU/ml in 88.6% for C. albicans, 84.8% for C. dubliniensis, 91.6% for C krusei and 82.3% for C tropicalis. Despite of this, only laser radiation or methylene blue did not reduce significantly the number of CFU/ml of Candida samples, except for C tropicalis. It could be concluded that the photo activation of methylene blue by the red laser radiation at 685 nm presented fungicide effect on all Candida species studied. (c) 2006 Elsevier B.V. All rights reserved.

Effect of sodium hypochlorite and five intracanal medications on Candida albicans in root canals

The aim of this study was to evaluate the effect of 1% sodium hypochlorite and five intracanals medications on Candida albicans harvested inside root canals. The contaminated canals were irrigated with sterile saline solution and then treated as follows: (i) filled with Calen paste (calcium hydroxide/glycol polyethylene paste); (ii) filled with camphorated paramonochloro phenol (CPMC); (iii) filled with 2% iodine-iodate solution; (iv) filled with tricresol formalin; (v) filled with Calen and CPMC pastes; (vi) irrigation with 1% sodium hypochlorite and filled with no intracanal medication; and (vii) no intracanal medication was used. Canal access and the apical foramen were then sealed with Cavit and the roots were stored in a humid chamber at 37 +/- 1 degreesC for 14 days. The canals were reinstrumented and irrigated with sterile saline solution. Sterile paper points were used to transfer the root canal contents to test tubes containing sterile saline solution. Part of the suspension was harvested in Sabouraud dextrose agar with chloramphenicol and incubated at 37 +/- 1 degreesC for 48 h, CPMC was effective in 100% of the samples followed in decreasing order of effectiveness by calcium hydroxide with CPMC (70% effective), 1% sodium hypochlorite (70% effective) (p < 0.05), tricresol formalin (60% effective), 2% iodine-iodate solution (50% effective), calcium hydroxide paste (30% effective), and saline + no intracanal medication.