Blood and intestinal parasites of squirrels (Rodentia: Sciuridae) in Amazonian Brazil

We report the result of an examination for blood and intestinal protozoa in 12 specimens of the red squirrel Sciurus spadiceus (Rodentia: Sciuridae) from Birroque, municipality of Plácido de Castro, state of Acre, Brazil. No parasites were detected in thin, Giemsa-stained blood films of the animals, but culture of the blood of three in Difco B45 medium blood-agar slants gave rise to isolates of epimastigotes. Inoculation of one isolate into laboratory mice resulted in the appearance of Trypanosoma cruzi-like trypomastigotes in their peripheral blood, and the other two isolates gave rise to transient infections with a T. lewisi-like parasite in inoculated mice and hamsters. The failure of the latter parasite to develop in the triatomine bug Rhodnius robustus suggests that it is probably not T. rangeli. This appears to be the first record of a T. lewisi-like trypanosome in neotropical squirrels. Oocysts of an Eimeria sp., were detected in the faeces of 10 animals (83.3%). The parasite develops in the epithelial cells of the intestine, where it may cause severe damage and sometimes results in death of the animal. No oocysts were detected in bile.

Antifungal activity from Ocimum gratissimum L. towards Cryptococcus neoformans

Cryptococcal infection had an increased incidence in last years due to the explosion of acquired immune deficiency syndrome epidemic and by using new and effective immunosuppressive agents. The currently antifungal therapies used such as amphotericin B, fluconazole, and itraconazole have certain limitations due to side effects and emergence of resistant strains. So, a permanent search to find new drugs for cryptococcosis treatment is essential. Ocimum gratissimum, plant known as alfavaca (Labiatae family), has been reported earlier with in vitro activity against some bacteria and dermatophytes. In our work, we study the in vitro activity of the ethanolic crude extract, ethyl acetate, hexane, and chloroformic fractions, essential oil, and eugenol of O. gratissimum using an agar dilution susceptibility method towards 25 isolates of Cryptococcus neoformans. All the extracts of O. gratissimum studied showed activity in vitro towards C. neoformans. Based on the minimal inhibitory concentration values the most significant results were obtained with chloroformic fraction and eugenol. It was observed that chloroformic fraction inhibited 23 isolates (92%) of C. neoformans at a concentration of 62.5 µg/ml and eugenol inhibited 4 isolates (16%) at a concentration of 0.9 µg/ml. This screening may be the basis for the study of O. gratissimum as a possible antifungal agent.

Value of morphotyping for the characterization of Candida albicans clinical isolates

Until recently, morphotyping, a method evaluating fringe and surface characteristics of streak colonies grown on malt agar, has been recommended as a simple and unexpensive typing method for Candida albicans isolates. The discriminatory power and reproducibility of Hunter's modified scheme of Phongpaichit's morphotyping has been evaluated on 28 C. albicans isolates recovered from the oral cavity of asymptomatic human immunodeficiency virus-positive subjects, and compared to two molecular typing methods: randomly amplified polymorphic DNA (RAPD) fingerprinting, and contour clamped homogeneous electric field (CHEF) electrophoretic karyotyping. Morphological features of streak colonies allowed to distinguish 11 different morphotypes while RAPD fingerprinting yielded 25 different patterns and CHEF electrophoresis recognized 9 karyotypes. The discriminatory power calculated with the formula of Hunter and Gaston was 0.780 for morphotyping, 0.984 for RAPD fingerprinting, and 0.630 for karyotyping. Reproducibility was tested using 43 serial isolates from 15 subjects (2 to 6 isolates per subject) and by repeating the test after one year storage of the isolates. While genetic methods generally recognized a single type for all serial isolates from each of the subjects studied, morphotyping detected strain variations in five subjects in the absence of genetic confirmation. Poor reproducibility was demonstrated repeating morphotyping after one year storage of the isolates since differences in at least one character were detected in 92.9% of the strains.

Studies on antimicrobial activity, in vitro, of Physalis angulata L. (Solanaceae) fraction and physalin B bringing out the importance of assay determination

Complex physalin metabolites present in the capsules of the fruit of Physalis angulata L. have been isolated and submitted to a series of assays of antimicrobial activity against Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, S. aureus ATCC 25923, S. aureus ATCC 6538P, Neisseria gonorrhoeae ATCC 49226, Escherichia coli ATCC 8739; E. coli ATCC 25922, Candida albicans ATCC 10231 applying different methodologies such as: bioautography, dilution broth, dilution agar, and agar diffusion techniques. A mixture of physalins (pool) containing physalins B, D, F, G inhibit S. aureus ATCC 29213, S. aureus ATCC 25923, S. aureus ATCC 6538P, and N. gonorrhoeae ATCC 49226 at a concentration of 200 mg/µl, using agar dilution assays. The mixture was inactive against P. aeruginosa ATCC27853, E. coli ATCC 8739; E. coli ATCC 25922, C. albicans ATCC 10231 when applying bioautography assays. Physalin B (200 µg/ml) by the agar diffusion assay inhibited S. aureus ATCC 6538P by ± 85%; and may be considered responsible for the antimicrobial activity.

Phenotypic characterization of three clinical isolates of Burkholderia pseudomallei in Ceará, Brazil

Burkholderia pseudomallei, the causative agent of melioidosis was found in a small cluster of cases in Tejuçuoca, Ceará, Brazil. Tests were carried out to determine its phenotypic characteristics: colony morphology on Ashdown agar and MacConkey agar, biochemical profile in conventional biochemical tests and API 20NE, arabinose assimilation and susceptibility testing by disk diffusion, comparing with data in the literature. This study confirms the presence of B. pseudomallei in Brazil and describes its characteristics.

Synergism between plant extract and antimicrobial drugs used on Staphylococcus aureus diseases

Searches for substances with antimicrobial activity are frequent, and medicinal plants have been considered interesting by some researchers since they are frequently used in popular medicine as remedies for many infectious diseases. The aim of this study was to verify the synergism between 13 antimicrobial drugs and 8 plant extracts - "guaco" (Mikania glomerata), guava (Psidium guajava), clove (Syzygium aromaticum), garlic (Allium sativum), lemongrass (Cymbopogon citratus), ginger (Zingiber officinale), "carqueja" (Baccharis trimera), and mint (Mentha piperita) - against Staphylococcus aureus strains, and for this purpose, the disk method was the antimicrobial susceptibility test performed. Petri dishes were prepared with or without dilution of plant extracts at sub-inhibitory concentrations in Mueller-Hinton Agar (MHA), and the inhibitory zones were recorded in millimeters. In vitro anti-Staphylococcus aureus activities of the extracts were confirmed, and synergism was verified for all the extracts; clove, guava, and lemongrass presented the highest synergism rate with antimicrobial drugs, while ginger and garlic showed limited synergistic capacity.

Propionibacterium acnes: An Underestimated Pathogen in Implant-Associated Infections.

The role of Propionibacterium acnes in acne and in a wide range of inflammatory diseases is well established. However, P. acnes is also responsible for infections involving implants. Prolonged aerobic and anaerobic agar cultures for 14 days and broth cultures increase the detection rate. In this paper, we review the pathogenic role of P. acnes in implant-associated infections such as prosthetic joints, cardiac devices, breast implants, intraocular lenses, neurosurgical devices, and spine implants. The management of severe infections caused by P. acnes involves a combination of antimicrobial and surgical treatment (often removal of the device). Intravenous penicillin G and ceftriaxone are the first choice for serious infections, with vancomycin and daptomycin as alternatives, and amoxicillin, rifampicin, clindamycin, tetracycline, and levofloxacin for oral treatment. Sonication of explanted prosthetic material improves the diagnosis of implant-associated infections. Molecular methods may further increase the sensitivity of P. acnes detection. Coating of implants with antimicrobial substances could avoid or limit colonization of the surface and thereby reduce the risk of biofilm formation during severe infections. Our understanding of the role of P. acnes in human diseases will likely continue to increase as new associations and pathogenic mechanisms are discovered.

Antibacterial potentiality of Argemone mexicana solvent extracts against some pathogenic bacteria

The sensitivity of two Gram positive (Staphylococcus aureus and Bacillus subtilis) and two Gram negative (Escherichia coli and Pseudomonas aeruginosa) pathogenic multi-drug resistant bacteria was tested against the crude extracts (cold aqueous, hot aqueous, and methanol extracts) of leaves and seeds of Argemone mexicana L. (Papaveraceae) by agar well diffusion method. Though all the extracts were found effective, yet the methanol extract showed maximum inhibition against the test microorganisms followed by hot aqueous extract and cold aqueous extract.

Evaluation of MRSA-Screen, a simple anti-PBP 2a slide latex agglutination kit, for rapid detection of methicillin resistance in Staphylococcus aureus.

The MRSA-Screen test (Denka Seiken Co., Ltd., Tokyo, Japan), consisting of a slide latex agglutination kit that detects PBP 2a with a monoclonal antibody, was blindly compared to the oxacillin disk diffusion test, the oxacillin-salt agar screen, and PCR of the mecA gene for the detection of methicillin resistance in Staphylococcus aureus. A total of 120 methicillin-susceptible S. aureus (MSSA) and 80 methicillin-resistant S. aureus (MRSA) isolates, defined by the absence or presence of the mecA gene, respectively, were tested. The MRSA-Screen test, the oxacillin disk diffusion test, and the oxacillin-salt agar screening test showed sensitivities of 100, 61.3, and 82.5% and specificities of 99.2, 96.7, and 98.3%, respectively. We conclude that the MRSA-Screen is a very accurate, reliable, and fast test (15 min) for differentiation of MRSA from MSSA colonies on agar plates.

Slime production and antibiotic susceptibility in staphylococci isolated from clinical samples

A total of 187 isolates from several clinical specimens were identified to species level as 129 Staphylococcus aureus strains and 58 coagulase-negative staphylococci (CNS) strains by the API Staph System (Biomerieux). Slime production was detected both by the conventional Christensen's method as well as by the Congo red agar method. Seventy-two strains of staphylococci isolates (38.5%) were found to be slime producers by Christensen's test tube method whereas 58 strains (31%) were slime positive with Congo red agar method. There was no statistically significant difference between the two methods for the detection of slime production (P > 0.05). Susceptibility of isolates against antimicrobial agents was tested by the disk diffusion method. Staphylococcal species had resistance to one or more antibiotics. Among the various antimicrobial agents, oxacillin (71.1%) and erythromycin (47.1%) showed higher resistance than most of the agents used against all isolates. Oxacillin resistant S. aureus (ORSA) and oxacillin resistant coagulase-negative staphylococci (ORCNS), 97 (75.2%) and 36 (62.1%) respectively were frequently observed in strains isolated from clinical materials. Among the ORSA strains, two strains were resistant to vancomycin. Moreover, 96 (74.4%) of 129 S. aureus strains were positive for blactamase enzyme. However, 78 (81.25%) of 96 b-lactamase positive S. aureus strains were b-lactamase positive ORSA isolates, but none of them had vancomycin resistance.

In Vivo Systematic Analysis of Candida albicans Zn2-Cys6 Transcription Factors Mutants for Mice Organ Colonization.

The incidence of fungal infections in immuno-compromised patients increased considerably over the last 30 years. New treatments are therefore needed against pathogenic fungi. With Candida albicans as a model, study of host-fungal pathogen interactions might reveal new sources of therapies. Transcription factors (TF) are of interest since they integrate signals from the host environment and participate in an adapted microbial response. TFs of the Zn2-Cys6 class are specific to fungi and are important regulators of fungal metabolism. This work analyzed the importance of the C. albicans Zn2-Cys6 TF for mice kidney colonization. For this purpose, 77 Zn2-Cys6 TF mutants were screened in a systemic mice model of infection by pools of 10 mutants. We developed a simple barcoding strategy to specifically detect each mutant DNA from mice kidney by quantitative PCR. Among the 77 TF mutant strains tested, eight showed a decreased colonization including mutants for orf19.3405, orf19.255, orf19.5133, RGT1, UGA3, orf19.6182, SEF1 and orf19.2646, and four an increased colonization including mutants for orf19.4166, ZFU2, orf19.1685 and UPC2 as compared to the isogenic wild type strain. Our approach was validated by comparable results obtained with the same animal model using a single mutant and the revertant for an ORF (orf19.2646) with still unknown functions. In an attempt to identify putative involvement of such TFs in already known C. albicans virulence mechanisms, we determined their in vitro susceptibility to pH, heat and oxidative stresses, as well as ability to produce hyphae and invade agar. A poor correlation was found between in vitro and in vivo assays, thus suggesting that TFs needed for mice kidney colonization may involve still unknown mechanisms. This large-scale analysis of mice organ colonization by C. albicans can now be extended to other mutant libraries since our in vivo screening strategy can be adapted to any preexisting mutants.

In vitro activity of fosfomycin against extended-spectrum-beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae: comparison of susceptibility testing procedures

The agar dilution, broth microdilution, and disk diffusion methods were compared to determine the in vitro susceptibility of 428 extended-spectrum-beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae to fosfomycin. Fosfomycin showed very high activity against all ESBL-producing strains. Excellent agreement between the three susceptibility methods was found for E. coli, whereas marked discrepancies were observed for K. pneumoniae.

Ulcerative fungal keratitis in a Brown Swiss cow.

An 11-year-old Brown Swiss cow was referred to the Farm Animal Department of the Veterinary Teaching Hospital in Zurich, Switzerland, because of lateral recumbency due to puerperal hemolytic anemia. The animal had developed enophthalmos due to dehydration at the time of presentation. Two days after hospitalization, the cow showed blepharospasm and epiphora of the right eye. Ophthalmic examination of the right eye revealed a fluorescein-positive, paraxial, superficial corneal ulcer with focal edema, and mild superficial neovascularization. White corneal stromal infiltrates were seen at the edges of the ulcer bed. After initial topical treatment with an antibiotic ointment (Neomycin 3.5 mg/g, Bacitracin 250 IU/g) three times a day, an increase in corneal infiltrates was noted on re-examination 2 days later. Several fluorescein-negative, punctate, stromal, white opacities were seen dorsal to the ulcer. Cytology demonstrated the presence of fungal hyphae. Topical treatment with 2% miconazole ointment and 0.36% K-EDTA eye drops six times daily and four times daily, respectively, from the second day and continued antibiotics three times daily resolved the clinical symptoms within 6 days. Fungal culture identified the fungal organism as Eurotium amstelodami.

Specimen storage in transport medium and detection of group B streptococci by culture

Recovery of group B streptococci (GBS) was assessed in 1,204 vaginorectal swabs stored in Amies transport medium at 4 or 21°C for 1 to 4 days either by direct inoculation onto Granada agar (GA) or by culture in blood agar (BA) and GA after a selective broth enrichment (SBE) step. Following storage at 4°C, GBS detection in GA was not affected after 72 h by either direct inoculation or SBE; however, GBS were not detected after SBE in the BA subculture in some samples after 48 h of storage and in GA after 96 h. After storage at 21°C, loss of GBS-positive results was significant after 48 h by direct inoculation in GA and after 96 h by SBE and BA subculture; some GBS-positive samples were not detected after 24 h of storage followed by SBE and BA subculture or after 48 h of storage followed by SBE and GA subculture. Storage of swabs in transport medium, even at 4°C, produced after 24 h an underestimation of the intensity of GBS colonization in most specimens. These data indicate that viability of GBS is not fully preserved by storage of vaginorectal swabs in Amies transport medium, mainly if they are not stored under refrigeration

Pyoluteorin production by Pseudomonas fluorescens strain CHA0 is involved in the suppression of Pythium damping-off of cress but not of cucumber

Pseudomonas fluorescens strain CHA0 is an effective biocontrol agent of various soilborne pathogens. It controls damping-off or root rot caused byPythium ultimum on cucumber, wheat and cress. Strain CHA0 synthesizes several antibiotic metabolites such as hydrogen cyanide, 2,4-diacetylphloroglucinol, and pyoluteorin. The role of pyoluteorin in the suppression of damping-off was investigated. Two Tn5 mutants (CHA660 and CHA661) of strain CHA0 were isolated which had lost the capacity to produce pyoluteorin but still produced 2,4-diacteylphloroglucinol and HCN. These mutants still inhibitedP. ultimum on malt agar (which favours the production of 2,4-diacetylphloroglucinol) but had partially lost the ability to inhibit this pathogen on King's B agar (which favours the production of pyoluteorin). The two pyoluteorin-negative mutants showed a reduced capacity to suppress damping-off of cress caused byP. ultimum but were as effective in the protection of cucumber against this pathogen as the wild-type strain. These results indicate that, depending on the plant, pyoluteorin production plays a role in the suppression of damping-off by strain CHA0 without being a major mechanism in disease suppression. We suggest that the contribution of pyoluteorin to the biocontrol activity of strain CHA0 is determined by the quantity of this antibiotic produced in the rhizosphere, which might depend on the root exudates of the host plant.