Growth-promoting factors for yeast cells ofParacoccidioides brasiliensis

Low-density seedings of yeast cells ofParacoccidioides brasiliensis give poor growth (as assessed by plating efficiency test) on conventional mycological agar media, and therefore growth-promoting factors for this fungus were sought. Water-extracts of yeast cells of sixP. brasiliensis isolates were all considerably effective in promoting the growth of low-density seedings ofP. brasiliensis isolates Pb-18 and Hachisuga, but had little effect on isolate Bt-4. Horse serum, at a concentration range of 2–4%, moderately or considerably promoted the growth of theseP. brasiliensis isolates. Combinations of the fungus cell extracts with horse serum were highly effective in promoting the growth of all of the fungal isolates. The fungus cell extracts showed siderophore (microbial iron carrier) activity. An iron-chelator, ethylenediaminetetraacetic acid, at a concentration of 100 μM also highly promoted the growth of the fungal isolates in the presence of horse serum, and ferric ion added to culture medium was considerably effective in the growth promotion. These results suggest that deficient utilization of external iron by the fungus cell is one of the growth-limiting processes for low-density seedings of yeast cells ofP. brasiliensis on conventional mycological agar media.     

Paracoccidioides brasiliensis isolated from armadillos is virulent to Syrian hamsters

Isolates of Paracoccidioides brasiliensis may vary in virulence according to time of in vitro subcultivation. The present study compared the morphology and pathogenicity to hamsters of two P. brasiliensis isolates: one obtained from human lesions and maintained in the laboratory for several years (Pb-18) and the other isolate recovered from hamsters inoculated with organ homogenates from armadillos (Pb-T). The microscopic morphology of Pb-18 and Pb-T showed yeast cells with similar diameter. However, Pb-T produced a significantly higher number of buds per mother cell than Pb-18. Besides, the mycelial form of Pb-T developed abundant sporulation during 8 weeks of culture which was absent in the Pb-18 isolate. Virulence studies demonstrated that mortality rates, antibody levels, fungal load and extent of lesions in the organs were significantly higher in animals infected with Pb-T. The results demonstrated that Pb-T recently isolated from an animal was more virulent than Pb-18. These differences between the two P. brasillensis isolates may be indicators of virulence attenuation in this fungal species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Granuloma formation and killing functions of granuloma in congenitally athymic nude mice infected with Blastomyces dermatitidis and Paracoccidioides brasiliensis

We did this experiment to clarify the mechanism of granuloma formation and the killing functions of granuloma in nude mice against Blastomyces dermatitidis and Paracoccidioides brasiliensis infections. B. dermatitidis A-295 and P. brasiliensis B-1183 were the cultures used. Congenitally athymic nude (nu/ nu) mice and their heterozygous (nu/ +) littermates of BALB/ c background were the test animals. From culture A-295, 0.1% and 1% cell suspensions (wet weight) were prepared and from culture B-1183 0.2% and 2% cells suspensions were prepared. Ten nu/ + and 10 nu/ nu mice were allotted to each of four cell suspensions. For experimental blastomycosis each mouse was inoculated intravenously with 0.2 ml of the cell suspension of A-295 and for experimental paracoccidioidomycosis, with 0.15 ml of the cell suspension of B-1183. Two mice from each of the four groups were killed at 5, 8, 12, 18 and 25 days after inoculation, and histopathologic sections, stained with H&E or by PAS, were prepared from various internal organs.In the nu/ nu mice inoculated with B. dermatitidis A-295 granuloma was formed in the brain tissue after the 12th day. However, mononuclear cells, which formed the granuloma, did not kill the fungal cells, and the fungal cells continued to multiply in the granuloma. On the other hand, in the heart, kidney and fat tissue, their histopathological findings after the 18th day were clumps of fungal cells with slight cell reactions. In these organs the exertion of cell-mediated immunity was necessary for granuloma formation against the fungal infection.In the nu/ nu mice infected with P. brasiliensis B-1183, granuloma appeared in the brain and kidney after the 18th day and fungal cells continued to multiply within the granuloma as well as in those inoculated with culture A-295.These results show that the exertion of cell-mediated immunity plays an important role as the defense mechanisms of hosts against these fungal infections. However, PMNs also play an important role in the mouse's defense mechanisms against these fungal infections.We assume that the defense mechanisms of immunocompetent mice against B. dermatitidis or P. brasiliensis infection consist chiefly of two steps: in the first step phagocytosis by PMNs occurs and in the second step cell-mediated immunity enters into play.     

Mitochondrial protein synthesis in aerobic and micro-aerobic continuous cultures ofCandida parapsilosis

Candida parapsilosis grows oxidatively under aerobic conditions and fermentatively in micro-aerobic (0.2 M oxygen) continuous culture. The amount of unsaturated fatty acids and sterol in cells and mitochondria, and the aerobic cytochromes are decreased in micro-aerobic cultures. In aerobic cells mitochondrial protein synthesis accounts for the formation of more than one-third of the proteins of the organelles; in micro-aerobic cultures, this declines to 5%. At the same time, minor but significant differences in the nature of the protein products formed by the mitochondrial system are evident in micro-aerobic and aerobic cells.     

Stimulation of the growth of cutaneous strains of Peptococcus saccharolyticus by iron, haematin and blood

The growth of nine strains of Peptococcus saccharolyticus was assessed quantitatively by culture Trypticase Soy/yeast extract/Tween 80 agar (TSY-TW) with and without supplementation with iron or haematin and on blood agar, in aerobic, reduced 02 (3% O2 with 8% CO2, 8% H2 and 81% N2) and anaerobic atmospheres. All strains grew better anaerobically and under reduced O2 conditions than aerobically on supplemented or unsupplemented TSY-TW.Supplementation of TSY-TW with iron or haematin resulted in an average 4.4-fold increase in bacterial count in a reduced O2 atmosphere and an average 4.2-fold increase under anaerobic conditions. Under aerobic conditions the increase in count ranged from O to greater than 5000-fold, as some strains failed to grow on unsupplemented TSY-TW but responded well to the supplements of iron or haematin. The highest bacterial counts were obtained on Columbia blood agar incubated anaerobically. However, P. saccharolyticus failed to grow aerobically on plain or heated Columbia blood agar with or without supplements. TSY-TW blood agar supported the growth of the one strain tested under all three atmospheric conditions. The type strain (ATCC 14953) differed from all others in its failure to grow aerobically or in a reduced O2 atmosphere on supplement or unsupplemented media. Colony size varied greatly on different media, in different atmospheres and from strain to strain, being greatest in a reduced O2 atmosphere on Columbia blood agar. There was no correlation between the viable bacterial count and colony size.     

Isolation of amylolytic,xylanolytic, and cellulolytic microorganisms extracted from the gut of the termite Reticulitermes santonensis by means of a micro-aerobic atmosphere

The aim of this work was to isolate enzyme-producing microorganisms from the tract of the termite Reticulitermes santonensis. The microorganisms were extracted from the guts and anaerobic (CO₂ or CO₂/H₂) and micro-aerobic atmospheres were used to stimulate growth. Three different strategies were tried out. First, the sample was spread on Petri dishes containing solid media with carboxymethylcellulose, microcrystalline cellulose or cellobiose. This technique allowed us to isolate two bacteria: Streptomyces sp. strain ABGxAviA1 and Pseudomonas sp. strain ABGxCellA. The second strategy consisted in inoculating a specific liquid medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. The samples were then spread on Petri dishes with the same specific medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. This led to the isolation of the mold Aspergillus sp. strain ABGxAviA2. Finally, the third strategy consisted in heating the first culture and spreading samples on agar plates containing rich medium. This led to the isolation of the bacterium Bacillus subtilis strain ABGx. All those steps were achieved in controlled atmospheres. The four enzyme-producing strains which were isolated were obtained by using a micro-aerobic atmosphere. Later, enzymatic assays were performed on the four strains. Streptomyces sp. strain ABGxAviA1 was found to produce only amylase, while Pseudomonas sp. strain ABGxCellA was found to produce β-glucosidase as well. Aspergillus sp. strain ABGxAviA2 showed β-glucosidase, amylase, cellulase, and xylanase activities. Finally, B. subtilis strain ABGx produced xylanase and amylase.     

Effect of Modified Atmosphere Composition on the Metabolism of Glucose by Brochothrix thermosphacta

The influence of atmosphere composition on the metabolism of Brochothrix thermosphacta was studied by analyzing the consumption of glucose and the production of ethanol, acetic and lactic acids, acetaldehyde, and diacetyl-acetoin under atmospheres containing different combinations of carbon dioxide and oxygen. When glucose was metabolized under oxygen-free atmospheres, lactic acid was one of the main end products, while under atmospheres rich in oxygen mainly acetoin-diacetyl was produced. The proportions of the total consumed glucose used for the production of acetoin (aerobic metabolism) and lactic acid (anaerobic metabolism) were used to decide whether aerobic or anaerobic metabolism predominated at a given atmosphere composition. The boundary conditions between dominantly anaerobic and aerobic metabolisms were determined by logistic regression. The metabolism of glucose by B. thermosphacta was influenced not only by the oxygen content of the atmosphere but also by the carbon dioxide content. At high CO₂ percentages, glucose metabolism remained anaerobic under greater oxygen contents.     

An ultrastructural comparison of the cell envelopes of selected strains of Nocardia asteroides and Nocardia brasiliensis

Growth curves were determined for three strains each ofNocardia asteroides andNocardia brasiliensis. Two strains ofN. brasiliensis and one strain ofN. asteroides had longer lag periods of growth than the remaining three strains. All strains had generation times of approximately 5.5 hours.The ultrastructure of the cell envelope of eachNocardia strain in early stationary phase growth was also examined. All the strains had typical trilaminar cell walls and cell membranes. The thickness of the cell wall layers, especially the inner peptidoglycan layer, varied from strain to strain. The inner layer of two strains ofN. brasiliensis and one strain ofN. asteroides was 12 nm or more in thickness, while that of the remaining three strains was 7 nm thick. These observed differences in growth patterns and/or thickness of the cell wall layers could be correlated to the varying degress of virulence as well as the divergent pathologies exhibited by these organisms.     

Nutritional studies on a methionine-requiring strain of Paracoccidioides brasiliensis

Strain IVIC-Pb9, unlike other strains ofParacoccidioides brasiliensis, cannot grow on a simple basal medium and requires the addition of casein hydrolyzate or yeast extract. The present study shows that this requirement is limited to very low concentrations of methionine and that methionine concentrations above 0.01% inhibit growth. The levels of glucose and organic nitrogen required for maximum rate of growth of strain IVIC-Pb9 on both basal medium and GGY medium composed of glucose, glycine and yeast extract were also determined. An evaluation of the suitability of the GGY medium revealed that its composition, as commonly used to grow dimorphic fungi, is not adequate to obtain a maximum rate of growth with strain IVIC-Pb9 ofP. brasiliensis.     

The effect of Lactobacillus buchneri and L. plantarum, applied at ensiling, on the ensiling fermentation and aerobic stability of wheat and sorghum silages

The effect of applying Lactobacillus buchneri (LB), alone or in combinations with L. plantarum (LP) and yeasts at ensiling, on the ensiling fermentation and aerobic stability of wheat and sorghum silages was studied under laboratory conditions. Treatments comprised LB, LP, yeasts, LB + yeasts, LP + yeasts, LB + LP and B-589 (a lactic acid bacterial strain isolated from wheat silage in Israel) alone. The treatments were also applied to sterilized aqueous extracts of wheat which were incubated at 30°C for 10 days. The pH of all treatments was below 4.0 already on day 4 of the experiment. Silages treated with LB had higher acetic acid concentrations than those treated with LP: 32–34 vs 16–18, and 28–34 vs 4–7 g kg⁻¹ in the experiments with wheat and sorghum, respectively. Similar results were obtained in wheat extracts. In the aqueous phase, marked differences in pH decrease were noticed among the treatments: 4.4 in LB, 6.0 in the yeast, and 3.7 in LP and B-589 (from day 3 and onwards). In both crops LB resulted in aerobically stable silages when applied alone or with LP and yeasts, whereas LP resulted in unstable silages upon aerobic exposure; the stability of the LB-treated silages is attributed to the higher acetic acid concentrations. The isolated strain (B-589) did not exhibit any advantage with regard to aerobic stability. Received 26 April 1999/ Accepted in revised form 05 July 1999     

Denitrification under various aeration conditions in Comamonas sp., strain SGLY2

Abstract A number of bacteria were isolated from different anoxic reactors. Those having denitrifying potential were tested for their ability to denitrify under aerobic conditions. The activity of their denitrifying enzymes varied from partial inactivation by oxygen (strains NO2B9 and TCET1) to oxygen-independent activity in a strain named SGLY2 which was tentatively identified as Comamonas sp. The effect of different aeration conditions on growth and on denitrification of SGLY2 was studied more extensively. This strain was able to consume oxygen and nitrate simultaneously with the production of nitrogen and without build-up of nitrite. The dissimilatory nitrate-reductase of nitrate-adapted cells was found to be more active in the presence of oxygen than in micro-aerobic or strictly anaerobic conditions.     

Effect of modified atmosphere composition on the metabolism of glucose by Brochothrix thermosphacta

The influence of atmosphere composition on the metabolism of Brochothrix thermosphacta was studied by analyzing the consumption of glucose and the production of ethanol, acetic and lactic acids, acetaldehyde, and diacetyl-acetoin under atmospheres containing different combinations of carbon dioxide and oxygen. When glucose was metabolized under oxygen-free atmospheres, lactic acid was one of the main end products, while under atmospheres rich in oxygen mainly acetoin-diacetyl was produced. The proportions of the total consumed glucose used for the production of acetoin (aerobic metabolism) and lactic acid (anaerobic metabolism) were used to decide whether aerobic or anaerobic metabolism predominated at a given atmosphere composition. The boundary conditions between dominantly anaerobic and aerobic metabolisms were determined by logistic regression. The metabolism of glucose by B. thermosphacta was influenced not only by the oxygen content of the atmosphere but also by the carbon dioxide content. At high CO(2) percentages, glucose metabolism remained anaerobic under greater oxygen contents.     

Morphological, Biochemical and Molecular Approaches for Comparing Typical and Atypical Paracoccidioides brasiliensis Strains

We evaluated the morphology of typical and atypical Paracoccidioides brasiliensis strains and the expression of its 43 kDa glycoprotein (GP43). Strains of P. brasiliensis preserved under mineral oil for long periods of time presented different morphological patterns on peptone, yeast-extract and glucose (PYG) agar. The intravenous inoculation in BALB/c mice confirmed that a strain bearing morphological alterations was non-virulent. In contrast, another strain also maintained under mineral oil but which did not exhibit such morphological dysfunction was as virulent as the well characterized Pb 339 and Pb 18 strains. The expression of the main antigen expressed by P. brasiliensis, GP43, was assessed in culture filtrates by western immunoblots. Typical and atypical strains were capable of secreting the glycoprotein, except strain Pb IOC 1059. The identity of the atypical strains was confirmed by PCR using specific primers for gp43, though the single PCR-fragment varied in size for the atypical strains. The PCR fragments from an atypical strain, Pb IOC 1210, and the typical Pb 339 and Pb IOC 3698 strains were sequenced and blasted to the gp43 gene from the Pb 18 strain (GenBank AY005429). These results ensured the identity of the atypical strains as P. brasiliensis, and suggested a relationship between the alteration of morphological differentiation and the virulence factor following storage under mineral oil.     

Growth of Pseudomonas fluorescens in modified atmosphere packaged tofu

Aims: The objective was to study the growth of Pseudomonas in a food product (tofu) where it typically occurs as a spoilage organism, and when this product is stored under modified atmosphere. Methods and Results: A Pseudomonas strain was isolated from the endogenous microflora of tofu. Tofu was inoculated with the strain, packaged in different gas conditions (air, 100% N₂, 30% CO₂/70% N₂ or 100% CO₂) and stored under refrigerated conditions. Microbial loads and the headspace gas composition were monitored during storage. Conclusions: The strain was capable of growing in atmospheres containing no or limited amounts of oxygen and increased amounts of carbon dioxide. Even when 100% CO₂ was used, growth could not be inhibited completely. Significance and Impact of Study: In contrast to the general characteristics of the genus Pseudomonas (strictly aerobic, highly sensitive to CO₂), it should not be expected in the food industry that removing oxygen from the food package and increasing the carbon dioxide content, combined with cold storage, will easily avoid spoilage by Pseudomonas species. Guarantee of hygienic standards and combination of strategies with other microbial growth inhibiting measures should be implemented.     

<Emphasis Type="Italic">Chromocurvus halotolerans</Emphasis> gen. nov., sp. nov., a gammaproteobacterial obligately aerobic anoxygenic phototroph,isolated from a Canadian hypersaline spring

A strain EG19^T of aerobic bacteria able to form pleomorphic cells was isolated from a brine spring runoff stream in the west central region of the province of Manitoba, Canada. The pale pinkish purple strain contained bacteriochlorophyll a incorporated into light-harvesting I and reaction center complexes. Its inability to grow under anaerobic illuminated conditions prompted designation as a member of the functional group known as aerobic anoxygenic phototrophic bacteria. Phylogenetic analysis of the 16S rRNA gene sequence revealed that it belonged to the Gammaproteobacteria, forming a distinct branch of phototrophs distantly related to most described aerobic anoxygenic phototrophs, quite marginally related (95.6%) both to the only other described gammaproteobacterial aerobic phototroph, Congregibacter litoralis, and also to nonphototrophs in the genus Haliea (95.1–96.1%). Physiological tests demonstrated tolerance profiles to salinity (0–18% NaCl), pH (7–12), and temperature (7–40°C) consistent with survival in a shallow hypersaline stream on the exposed, vegetation-depleted salt playa of its native East German Creek. Phylogenetic data and phenotypic properties such as pigment composition, morphology, and physiology support the proposal of the novel genus and species Chromocurvus halotolerans gen. nov., sp. nov., with EG19^T (=DSM 23344^T, =VKM B-2659^T) as the type strain.     

Metabolic engineering of Bacillus subtilis for enhanced production of acetoin

Acetoin is widely used in food and other industries. A bdhA and acoA double-knockout strain of Bacillus subtilis produced acetoin at 0.72?mol/mol, a 16.4?% increased compared to the wild type. Subsequent overexpression of the alsSD operon enhanced the acetolactate synthase activity by 52 and 66?% in growth and stationary phases, respectively. However, deletion of pta gene caused little increase of acetoin production. For acetoin production by the final engineered strain, BSUW06, acetoin productivity was improved from 0.087?g/l?h, using M9 medium plus 30?g glucose/l under micro-aerobic conditions, to 0.273?g/h?l using LB medium plus 50?g glucose/l under aerobic conditions. In fermentor culture, BSUW06 produced acetoin up to 20?g/l.     

In vivo and in vitro characteristics of six Paracoccidioides brasiliensis strains

The yeast-like forms of six P. brasiliensis strains were characterized and compared using in vitro (growth curve determination) and in vivo (pathogenicity to sensitive inbred mice) criteria. Strains Pb 18 and Pb 265 which behaved similarly in vitro, showing low counts of fungi and long mean generation times, were respectively the most and the least pathogenic strains. Strains Pb 2052 and IVIC Pb 267, which grow abundantly in vitro were, respectively virulent and avirulent. Strains Pb SN and IVIC Pb 9 behaved similarly both in vitro and in vivo displaying an intermediate pattern of virulence and growing conditions.     

Alterations in the pathogenicity of oneParacoccidioides brasiliensis isolate do not correlative with itsin vitro growth

Thein vitro subcultivation of some microorganisms for long periods causes measurable loss of their pathogenicity, which can be reverted by reisolation from infected hosts. We compared the pathogenicity and thein vitro growth pattern of oneP. brasiliensis isolate (Pb 18) in its yeast phase, using the following samples: 1) The original pathogenic Pb 18 (OP). 2) Pb 18 attenuated by continuousin vitro subcultivation (AT). 3) Pb 18 (AT) reisolated from susceptible B 10.A mice (RS). 4) Pb 18 (AT) reisolated from resistant A/SN mice (RR). Pathogenicity was evaluated by anatomopathology and mortality of mice infected i.p. with 5×10⁶ fungi. Median survival times of mice infected with OP ranged from 74 to 117 days during the first 51 months of subculturing; with more cycles of subculturing the median survival time increased, reaching 250 days at the 64^th month. This indicated decreasing virulence of OP during this period of subculturing. Survival of mice infected with RS and RR was respectively 112 and 123 days, which is similar to the behavior of the OP variant. Thein vitro growth curve profile of RR showed significantly higher numbers of total and viable yeasts than the other studied variant. These results show that: 1) Pb 18 isolate loses its pathogenicity by continuous subcultivation. This phenomenon is reverted by reisolation from mice, independently from their susceptibility to the fungus; 2) thein vitro growth patterns of Pb 18 do not correlate with alterations in pathogenicity but are influenced by the host's environment.     

Characteristics of 17Paracoccidioides brasiliensis isolates

We have studied the physiological and morphological features of 17 isolates ofParacoccidioides brasiliensis in order to define their phenotypes. The isolates were cultured at room temperature on potato dextrose agar (PDA, Difco) slants for mycelial growth and in 1% dextrose brain heart infusion agar (BHIA, Difco) at 37°C for the study of yeast forms. Most mycelial and yeast forms grew well between pH 5.6–9.4. In their response to osmotic pressure the isolates were separated in three groups: intolerant, intermediate and tolerant. They also varied in carbohydrate assimilation tests, which indicated important metabolic variation. No clear differences were observed in phenol oxidase tests, KNO₃, starch, casein and arbutin assimilation tests. Only 1 of the isolates, Bt-19, had gelatinase activity. No correlation was observed between the above differences and virulence. Two patterns of growth were observed in the mycelial cultures, glabrous and cottonous, the latter being correlated with increased virulence for ddY mice. Most yeast forms grew as cerebriform colonies, but Pb-HC and Bt-19 colonies had a cobblestone-like surface.