Experimental Pathogenicity of a Clinical Isolate of <Emphasis Type="Italic">Trichosporon dermatis</Emphasis> in a Murine Model

The pathogenicity of Trichosporon dermatis isolated from skin lesions of a patient has been examined in mice. Balb/c mice were treated with two intraperitoneal injections of 100 mg/kg cyclophosphamide on days 4 and 1 and one subcutaneous injection of 10 mg/kg dexamethasone on day 1 pre-inoculation, and then challenged with 0.2 ml T. dermatis inoculum (1 × 10⁸ CFU/ml) by topical application on an abrasive wound in the dermabrasive group and by hypodermic injection in the subcutaneous group. In the intravenous group, 0.2 ml of high (1 × 10⁸ CFU/ml) or low (1 × 10⁷ CFU/ml) inoculum was injected into the tail vein. Histopathology and inverse fungal culture were performed on the skin lesion and viscera, and renal fungal burden was also determined. Inoculated sites developed localized infections after dermabrasive and subcutaneous challenge in all mice, but the maximum area of skin lesions, and number of positive cultures from the lesions, were higher for immunocompromised mice. In the intravenous group, all immunocompetent animals survived during the four-week period, whereas 100 and 70% of immunocompromised animals died by 3 and 5 days in the high and low-inoculum groups, respectively. The incidence of disseminated infection and the renal fungal burden of immunocompromised mice were higher than those of immunocompetent mice. Our results demonstrate that subcutaneous and intravenous injection of T. dermatis can successfully establish cutaneous and systemic infection models in immunocompromised mice, with the kidney and lung being most susceptible.     

Local production of CCL3, CCL11, and IFN-γ correlates with disease severity in murine parainfluenza virus infection

Background

Using a murine model of parainfluenza virus infection (mPIV1 or Sendai virus; SeV), we compared the inflammatory responses to lethal and sub-lethal infections in inbred DBA/2 mice.

Methods

Mice were intranasally inoculated with either 1.6×10³ or 1.6×10⁵ infectious units (IU) of SeV or diluent control. Clinical data including daily weights, oxygen saturation, and lung function via whole body plethysmography were collected on days 0, 3–7, and 9–14. Clarified whole lung homogenates were evaluated for inflammatory markers by enzyme-linked immunoassay (ELISA). Data were analyzed using ANOVA or Student t-tests, as appropriate.

Results

Mice inoculated with 1.6×10⁵ IU of SeV developed a lethal infection with 100% mortality by day 7, while mice inoculated with 1.6×10³ IU developed a clinically significant infection, with universal weight loss but only 32% mortality. Interestingly, peak virus recovery from the lungs of mice inoculated with 1.6×10⁵ IU of SeV did not differ substantially from that detected in mice that received the 100-fold lower inoculum. In contrast, concentrations of CCL5 (RANTES), CCL11 (eotaxin), interferon-γ, CXCL10 (IP-10), and CCL3 (MIP-1α) were significantly higher in lung tissue homogenates from mice inoculated with 1.6×10⁵ IU (p?<?0.05). In the lethal infection, levels of CCL11, interferon- γ and CCL3 all correlated strongly with disease severity.

Conclusion

We observed that severity of SeV-infection in DBA/2 mice was not associated with virus recovery but rather with the levels of proinflammatory cytokines, specifically CCL11, interferon- γ and CCL3, detected in lung tissue in response to SeV infection.

     

Oral Administration of Live <Emphasis Type="Italic">Bifidobacterium</Emphasis> Substrains Isolated from Centenarians Enhances Intestinal Function in Mice

We studied the effects of two bifidobacteria strains isolated from healthy centenarians on intestinal function in mice. Bifidobacterium adolescentis BBMN23 and Bifidobacterium longum BBMN68 were orally administrated to specific pathogen-free BALB/c mice at different doses (2 × 10¹¹, 2 × 10⁹, or 2 × 10⁷ CFU/kg body weight) each day for 4 weeks. Villus height, crypt depth, villus width, and villus/crypt ratio (V/C) were determined. The content of duodenal secreted immunoglobulin A (sIgA) was also evaluated. There were clear increases in height and width of duodenal villi in both treated groups. Crypt depths were deeper in animals treated with BBMN23 than in controls, while depths were reduced in animals receiving BBMN68. The V/C ratio was increased after feeding with BBMN68, while BBMN23 had no significant effect. Both strains improved the sIgA content of the duodenum. These results suggest that BBMN23 and BBMN68 may improve intestinal digestion and ability and enhance immune barrier function in the intestine.     

Evaluation of DNA extraction methods for Bacillus anthracis spores isolated from spiked food samples

Aims

Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10‐fold serial dilutions of Bacillus anthracis spores using quantitative real‐time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Positive; MoBio PowerFood; ABI PrepSeq) were subsequently tested for their ability to isolate DNA from trace amounts of B. anthracis spores (approx. 6·5 × 10¹ and 1·3 × 10² CFU in 25 ml or 50 g of food sample) spiked into complex food samples including apple juice, ham, whole milk and bagged salad and recovered with immunomagnetic separation (IMS).

Methods and Results

The MasterPure kit effectively and consistently isolated DNA from low amounts of B. anthracis spores captured from food samples. Detection was achieved from apple juice, ham, whole milk and bagged salad from as few as 65 ± 14, 68 ± 8, 66 ± 4 and 52 ± 16 CFU, respectively, and IMS samples were demonstrated to be free of PCR inhibitors.

Conclusions

Detection of B. anthracis spores isolated from food by IMS differs substantially between commercial DNA extraction kits; however, sensitive results can be obtained with the MasterPure Gram Positive kit.

Significance and Impact of the Study

The extraction protocol identified herein combined with IMS is novel for B. anthracis and allows detection of low levels of B. anthracis spores from contaminated food samples.     

Virulence of <Emphasis Type="Italic">Sporothrix luriei</Emphasis> in a Murine Model of Disseminated Infection

Sporothrix luriei is a rare fungus causing sporotrichosis in humans. The virulence of this fungus was evaluated in a murine model of disseminated infection. Mice were challenged intravenously with two different inocula (2 × 10⁵ and 2 × 10⁷ CFU/animals) but only the highest one was able to kill the animals. Infected mice died between days 12 and 16, liver and spleen being the most affected organs. In the infected tissues, a massive infiltration of fungal cells and phagocytes were observed, but not the typical “eyeglass” cells described in infected human tissue.     

Development of a real-time TaqMan assay to detect <Emphasis Type="Italic">mendocina</Emphasis> sublineage <Emphasis Type="Italic">Pseudomonas</Emphasis> species in contaminated metalworking fluids

A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but are also time-consuming. The primer–probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 10¹ colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer–probe pair was successful in determining concentrations from used MWF samples, indicating levels between 2.9 × 10³ and 3.9 × 10⁶ CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 × 10¹ to 1.4 × 10⁵ CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer–probe pair can be efficiently used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs.     

Immunological responses induced by <Emphasis Type="Italic">asd</Emphasis> and <Emphasis Type="Italic">wzy/asd</Emphasis> mutant strains of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium in BALB/c mice

Attenuated bacteria have long been developed as vaccine candidates but can have some disadvantages, such as the potential for damage to immune organs due to insufficient clearance. To minimize these disadvantages, we generated Salmonella enterica serovar Typhimurium mutants SHJ2104 (asd::cm) and HTSaYA (wzy::km, asd::cm). The wzy gene codes for the O-antigen polymerase, which is involved in lipopolysaccharide (LPS) biosynthesis, and asd codes for aspartate β-semialdehyde dehydrogenase, which participates in cell wall formation. The strains synthesized LPS with a short-chain length, and showed lower cytotoxicity and reduced intracellular proliferation in animal cells compared to wild-type bacteria. After oral infection, the mutants were cleared in immune tissues, including the Peyer’s patch, mesenteric lymph node, and spleen, within 5 days. The LD50 of the mutants in Balb/c mice was estimated to be 10⁶ higher than wild-type bacteria when administered either via an oral or i.p. route, indicating that the two strains are highly attenuated. To compare the immune response to and protective effects of the mutants against wild-type bacterial infection, we inoculated the mutants into mice via an oral (1×10¹⁰CFU) or i.p. (1×10⁷ CFU) route once or twice at a two week interval. All immune responses, such as serum IgG and secretory IgA levels, cytokine production, and delayed hypersensitivity, were highly induced by two rounds of immunization. HTSaYA and SHJ2104 induced similar immune responses, and mice immunized with HTSaYA or SHJ2104 via an i.p. route were protected against wild-type Salmonella infection even at 100-fold of the LD₅₀ (5×10⁶ CFU). Taken together, these data indicate that HTSaYA and SHJ2104 could be developed as live attenuated Salmonella vaccine candidates.     

Beneficial activity of Enterococcus faecalis CECT7121 in the anti‐lymphoma protective response

Aims: To study the anti‐tumour effects of Enterococcus faecalis CECT7121 on LBC cells, an aggressive murine T‐cell lymphoma that kills the host in 18 days when is intraperitoneally (i.p.) administrated. Methods and Results: In vitro studies have shown that LBC cell proliferation was inhibited by Ent. faecalis CECT7121 stimulus in a dose‐dependent manner, inducing apoptosis. The production of ceramide was involved in the latter effect. To undertake in vivo studies, syngeneic BALB/c mice pre‐treated i.p. with Ent. faecalis CECT7121 (2·5 × 10⁸ CFU) were challenged i.p. with LBC cells (1·0 × 10⁶ cells) the day after. On day 30 post‐inoculation of LBC cells, 70% of Ent. faecalis CECT7121 pre‐treated mice survived, whereas no survivals were recorded in the control group. A group of surviving mice was re‐challenged with LBC cells, and 89% of them survived. Upon stimulation with irradiated LBC cells, spleen cell proliferation, high IFNγ, IL‐12 and IL‐10 levels were observed in surviving animals. Conclusions: Enterococcus faecalis CECT7121 affected multiple factors of the tumour establishment by the following methods: down‐regulating the LBC cell proliferation and inducing apoptosis in these cells; and enhancing the immune response that protects animals from lymphoma challenge and re‐challenge. Significance and Impact of the Study: This study demonstrate that Ent. faecalis CECT7121 has potential as a probiotic that could facilitate the development of novel complements to therapeutic strategies against oncological diseases.     

Carob pulp as raw material for production of the biocontrol agent <Emphasis Type="Italic">P. agglomerans</Emphasis> PBC-1

Large-scale production has been the major obstacle to the success of many biopesticides. The spreading of microbial biocontrol agents against postharvest disease, as a safe and environmentally friendly alternative to synthetic fungicides, is quite dependent on their industrial mass production from low-cost raw materials. Considerable interest has been shown in using agricultural waste products and by-products from food industry as nitrogen and carbon sources. In this work, carob pulp aqueous extracts were used as carbon source in the production of the biocontrol agent Pantoea agglomerans PBC-1. Optimal sugar extraction was achieved at a solid/liquid ratio of 1:10 (w/v), at 25°C, for 1 h. Batch experiments were performed in shake flasks, at different concentrations and in stirred reactors at two initial inoculums concentrations, 10⁶ and 10⁷ cfu ml⁻¹. The initial sugar concentration of 5 g l⁻¹ allowed rapid growth (0.16 h⁻¹) and high biomass productivity (0.28 g l⁻¹ h⁻¹) and was chosen as the value for use in stirred reactor experiments. After 22 and 32 h of fermentation the viable population reached was 3.2 × 10⁹ and 6.2 × 10⁹ cfu ml⁻¹ in the fermenter inoculated at 10⁶ cfu ml⁻¹ and 2.7 × 10⁹ and 6.7 × 10⁹ cfu ml⁻¹ in the bioreactor inoculated at 10⁷ cfu ml⁻¹. A 78% reduction of the pathogen incidence was achieved with PBC-1 at 1 × 10⁸ cfu ml⁻¹, grown in medium with carob extracts, on artificially wounded apples stored after 7 days at 25°C against P. expansum.     

Assessment of the Safety of Lactobacillus casei IMV B-7280 Probiotic Strain on a Mouse Model

Probiotics, in particular Lactobacillus (lactic acid bacteria, LAB) strains, are widely used in clinical practice. Despite that these probiotics have GRAS (generally regarded as safe) and qualified presumption of safety (QPS) statuses, the safety of particular strains still needs to be thoroughly studied. The aim of the study was to evaluate the safety of Lact. casei IMV B-7280 strain by investigating toxicity and the effects on gut microbiota in experimental animal model. Male BALB/c mice (7–8 weeks, weight 20–24 g) were treated with amounts of Lact. casei IMV B-7280 strain: 5 × 10⁶, 5 × 10⁸, or 5 × 10⁹ CFU/animal once per day during 7 days, or in the amount of 1 × 10¹⁰ CFU/animal once per day during 3 days (most of the proposed probiotic doses for humans—from 10⁸ to 10⁹ CFU) and monitored during 14 days. Blood tests and serum biochemistry were conducted; the cecal content from mice of the experimental and control groups were freshly collected and analyzed. At the end of the experiments (15th day), the presence of LAB in the heart, liver, kidney, and mesenteric lymph nodes and peripheral blood was determined; histology of the brain, liver, heart, fragments of the small and large intestine, and mesenteric lymph nodes was conducted. Survival rate of BALB/c mice treated with Lact. casei IMV B-7280 strain in different concentrations in toxicity experiments during 14 days was 100%. We observed no signs of toxicity as changes in gait, lethargy, sleep, somatomotor activity as well as changes in fur, eyes, skin and mucous membranes, tremors, behavior pattern, convulsions, salivation, diarrhea, and local injuries in mice from all experimental groups. After administration of probiotic strain, the number of opportunistic bacteria in cecal contents, such as Staphylococcus spp., Candida spp., Pseudomonas spp., and total aerobic and optionally anaerobic bacteria decreased compared to controls; the population of beneficial bacteria such as lactobacilli increased in cecal contents of these mice. LAB were not detected in the peripheral blood, heart, liver, kidneys, and mesenteric lymph nodes after administration of this strain to intact mice. Lact. casei IMV B-7280 strain is safe at dose up to 10¹⁰ CFU/animal during 3- and 7-day oral administration to mice and has a positive effect on the gut microbiota composition; it could be potentially considered as safe probiotic for humans.

     

Evaluation of a New Tumor Necrosis Factor-α-Inducing Membrane Protein of Helicobacter pylori as a Prophylactic Vaccine Antigen

Background: Tumor necrosis factor (TNF)‐α‐inducing protein (Tipα) is a newly identified carcinogenic factor present in Helicobacter pylori. Tipα has the unique function of inducing TNF‐α production by gastric cells in vitro and is assumed to be related with the development of gastritis and gastric cancer. We investigated the effects of vaccination with Tipα against H. pylori infection and analyzed the immune responses. Methods: C57BL/6 mice were immunized via the intranasal route with CpG, recombinant Tipα + CpG, and recombinant del‐Tipα (a mutant of Tipα) + CpG. Eight weeks after the mice were infected with H. pylori (5 × 10⁷ CFU), the number of colonizing bacteria in the stomach was calculated, and the histological severity of gastritis was evaluated. Levels of Tipα‐specific IgG and IgA antibodies in mouse serum were measured by an enzyme‐linked immunosorbent assay (ELISA). Local production of cytokines including Interleukin (IL)‐10, TNF‐α and Interferon (IFN)‐γ in gastric mucosa was also measured by real time‐PCR. Results: Levels of Tipα‐specific antibodies were significantly higher in Tipα‐immunized and del‐Tipα‐immunized mice than in the infection control group. The numbers of colonizing bacteria were significantly reduced in Tipα‐immunized mice (4.29 × 10⁵ CFU/g) and del‐Tipα immunized mice (2.5 × 10⁵ CFU/g) compared with infection control mice (5.7 × 10⁶ CFU/g). The levels of IFN‐γ and IL‐10 were significantly higher in del‐Tipα‐immunized mice than the infection control group. Conclusion: Vaccinations with Tipα and del‐Tipα were effective against H. pylori infection. The inhibition of H. pylori colonization is associated mainly with Th1 cell‐mediated immunity.     

Bioaerosol emissions in a poultry litter burning plant

This study investigates the exposure of workers to biological particles in a poultry litter burning plant in operation. The microorganism concentrations were examined at different workplaces during procedures leading to increased emissions. The concentrations of culturable airborne mesophilic, xerophilic and thermophilic microorganisms in the ambient air were tested inside and outside of the burning plant using two different methods of measuring. The focus of this study was on the quantitative evaluation of culturable bacteria as well as the quantitative and qualitative evaluation of gram-negative bacteria, fungi and thermophilic actinomycetes. The maximum airborne concentrations were found in the delivery hall. Mesophilic bacteria concentrations reached up to 1.7 × 10⁶ CFU/m³; gram-negative bacteria up to 9.1 × 10² CFU/m³. Fungal propagule concentrations for xerophilic fungi were between 1.2 × 10³ and 2.9 × 10⁴ CFU/m³ and for mesophilic fungi between 4.4 × 10² and 2.9 × 10⁴ CFU/m³. Among fungi, Aspergillus niger, Eurotium herbariorum and Scopulariopsis brevicaulis species were dominant. Thermophilic actinomycetes reached airborne concentrations of 8.7 × 10⁴ CFU/m³, with increased concentrations of the pathogens causing extrinsic allergic alveolitis. The high concentrations of airborne microorganisms in poultry litter burning plants and the potential hazard of the intake of microorganisms including potential pathogens require the introduction of consistent measures in both technical areas and personnel management.     

Airborne concentrations of bacteria and fungi in Thailand border market

Thailand border market is where the local Thais, Cambodians, Laotians, and Burmeses exchange their goods and culture at the border checkpoints. It is considered to be the source of aerial disease transmission especially for foreigners because it is always very crowded with people from all walks of life. Unhealthy air quality makes this area high risk of spread of airborne diseases. This study assessed airborne concentrations of bacteria and fungi in a border market to improve exposure estimates and develop efficient control strategies to reduce health risk. The density and distribution of airborne bacteria and fungi were investigated in the Chong Chom border market in Surin Province, Thailand. Eighteen air sampling sites were taken from outdoors and various work environments including indoor footpaths, wooden handicraft shops, electronic shops, the secondhand clothing shops, and fruit market areas. Exposed Petri plate method and liquid impinger sampler were used for sampling at the breathing zone, 1.5 m above the floor level, during weekend and holiday. Meteorological factors such as relative humidity, temperature, and light intensity were collected by portable data logger. The relative humidity was 67–73%, and temperature 29–33°C, and light varied between 18 and 270 Lux m⁻². Gram-positive and Gram-negative bacteria were found at a mean value of 10⁴ CFU m⁻³, and airborne fungi of 10³ CFU m⁻³ were recorded. The highest concentration of culturable airborne microorganisms was found along the indoor footpath (9.62 × 10⁴ CFU m⁻³ and 750.00 CFU/plate/h for impingement and sedimentation methods, respectively), the fruit market area (7.86 × 10⁴ CFU m⁻³ and 592.42 CFU/plate/h for impingement and sedimentation methods, respectively), and the secondhand clothing shop (4.59 × 10³ CFU m⁻³ and 335.42 CFU/plate/h for impingement and sedimentation methods, respectively) for Gram-positive bacteria, Gram-negative bacteria, and fungi, respectively. The lowest concentration of Gram-positive bacteria, Gram-negative bacteria, and fungi was found only at the outdoor area at 1.53 × 10⁴ CFU m⁻³, 0.93 × 10⁴ CFU m⁻³ and 0.80 × 10³ CFU m⁻³ by means of impingement method and 136.67 CFU/plate/h, 69.25 CFU/plate/h, and 62.00 CFU/plate/h by means of sedimentation methods for Gram-positive bacteria, Gram-negative bacteria, and fungi, respectively. The most frequently present airborne bacteria were identified as Bacillus, Corynebacteria, Diplococcus, Micrococcus, Acinetobacter, Alcaligenes, Enterobacter, and spore former rods. Acremonium, Aspergillus, Cladosporium, Penicillium, and Sporotrichum were the most frequently found aerosol fungi genera. The distribution of airborne microorganisms correlated with relative humidity and light factors based on principal component analysis. In conclusion, the border market is a potential source of aerial disease transmission and a various hazards of bioaerosols for workers, consumers, sellers, and tourists. The bioaerosol concentration exceeded the standard of occupational exposure limit. Many major indicators of allergenic and toxigenic airborne bacteria and fungi, Acinetobacter, Enterobacter, Pseudomonas, Cladosporium, Alternaria, Aspergillus, and Penicillium, were found in the various market environments.     

Quantification of airborne Aspergillus fumigatus spores in rabbit houses by real-time polymerase chain reaction

To accurately quantify airborne Aspergillus fumigatus (A. fumigatus) spores in rabbit houses, the real-time polymerase chain reaction (real-time PCR) and culture-based counting method (CCM) were employed to determine the airborne A. fumigatus spore concentrations. The results showed that, of the three rabbit houses (A, B, and C), the average concentrations of airborne A. fumigatus spores determined by real-time PCR were 3.0 × 10³, 3.3 × 10³, and 1.5 × 10³ spores/m³ air, respectively, while those determined by CCM were 2.5 × 10², 2.8 × 10², and 1.1 × 10² colony-forming unit/m³ air (CFU/m³ air), respectively, i.e., the former concentration was 12–14 times higher than the latter one. Therefore, the conventional CCM underestimated the concentrations of airborne fungal spores, and it is insufficient to determine the microbial aerosol concentration and evaluate the health risk only using CCM.     

Seasonal abundance and distribution of <Emphasis Type="Italic">Vibrio</Emphasis> species in the treated effluent of wastewater treatment facilities in suburban and urban communities of Eastern Cape Province,South Africa

We assessed the seasonal abundance and distribution of Vibrio species as well as some selected environmental parameters in the treated effluents of two wastewater treatment plants (WWTP), one each located in a suburban and urban community of Eastern Cape Province, South Africa. Vibrio population density ranged from 2.1×10⁵ to 4.36×10⁴ CFU/ml in the suburban community and from 2.80×10⁵ to 1.80×10⁵ CFU/ml in the urban community. Vibrio species associated with 180 μ, 60 μ, and 20 μ plankton sizes were observed at densities of 0–136×10³ CFU/ml, 0–8.40×10² CFU/ml, and 0–6.80×10² CFU/ml, respectively at the suburban community’s WWTP. In the urban community, observed densities of culturable Vibrio were 0–2.80×10² CFU/ml (180 μ), 0–6.60×10² CFU/ml (60 μm), and 0–1.80× 10³ CFU/ml (20 μm). The abundance of free-living Vibrio species ranged from 0 to 1.0×10² and 1.0×10³ CFU/ml in the suburban and urban communities’ WWTPs, respectively. Molecular confirmation of the presumptive Vibrio isolates revealed the presence of V. fluvialis (41.38%), V. vulnificus (34.48%), and V. parahaemolyticus (24.14%) in the suburban community effluents. In the urban community molecular confirmation revealed that the same species were present at slightly different percentages, V. fluvialis (40%), V. vulnificus (36%), and V. parahaemolyticus (24%). There was no significant correlation between Vibrio abundance and season, either as free-living or plankton-associated entities, but Vibrio species abundance was positively correlated with temperature (r=0.565; p<0.01), salinity, and dissolved oxygen (p<0.05). Turbidity and pH showed significant seasonal variation (p<0.05) across the seasons in both locations. This study underscores the potential of WWTPs to be sources of Vibrio pathogens in the watershed of suburban and urban communities in South Africa.     

Combination Efficacy of Voriconazole and Amphotericin B in the Experimental Disease in Immunodeficient Mice Caused by Fluconazole-resistant <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

The therapeutic efficacy of amphotericin B and voriconazole alone and in combination with one another were evaluated in immunodeficient mice (BALB/c-SCID) infected with a fluconazole-resistant strain of Cryptococcus neoformans var. grubii. The animals were infected intravenously with 3 × 10⁵ cells and intraperitoneally treated with amphotericin B (1.5 mg/kg/day) in combination with voriconazole (40 mg/kg/days). Treatment began 1 day after inoculation and continued for 7 and 15 days post-inoculation. The treatments were evaluated by survival curves and yeast quantification (CFUs) in brain and lung tissues. Treatments for 15 days significantly promoted the survival of the animals compared to the control groups. Our results indicated that amphotericin B was effective in assuring longest-term survival of infected animals, but these animals still harbored the highest CFU of C. neoformans in lungs and brain at the end of the experiment. Voriconazole was not as effective alone, but in combination with amphotericin B, it prolonged survival for the second-longest time period and provided the lowest colonization of target organs by the fungus. None of the treatments were effective in complete eradication of the fungus in mice lungs and brain at the end of the experiment.     

Endogenous Tumor Necrosis Factor (TNF) α Mediates Neutrophil Accumulation at the Mid-Phase of a Murine Model of Pseudomonas aeruginosa Pneumonia

To determine the role of endogenous tumor necrosis factor (TNF) α on neutrophil influx into the lungs in acute Pseudomonas aeruginosa pneumonia, we evaluated TNF α activity, inflammatory cell response and neutrophil chemotactic activity in the bronchoalveolar lavage fluids (BALFs) of P. aeruginosa-infected mice. In the case of fatal pneumonia, the TNF α activity in the BALFs appeared within 3 hr, peaked at 6–12 hr and attenuated within 24 hr after intratracheal challenging, while no TNF α activity was detected in the plasma. The elevation of TNF α activity in the BALFs was closely associated with neutrophil accumulation. Mirroring the TNF α activity response and the influx of neutrophils into the murine airway, the number of neutrophils in the BALFs increased within 3 hr, peaked at 6–12 hr and remained elevated up to 24 hr after challenging. Neutralization of the TNF α activity in the BALFs with anti-murine TNF antiserum decreased the level of neutrophil migration by BALF 45.0–49.7% at 6 hr and 49.3–54.2% at 12 hr, while the neutralizing antiserum had no effect on the level of neutrophil migration by BALFs at 3 and 24 hr. Furthermore, the intravenous administration of anti-murine TNF antiserum 2 hr before challenging significantly inhibited neutrophil migration into the lungs of mice with sublethal pneumonia (P < 0.05; compared with mice receiving pre-immune serum). These data suggest that intra-alveolar TNF α plays an important role in causing lung neutrophil accumulation at the mid-phase of murine P. aeruginosa pneumonia.     

Air biocontamination in a variety of agricultural industry environments in Egypt: a pilot study

Inhalation of airborne microorganisms and organic dust is an occupational concern among workers in agricultural industries. Airborne microorganisms and particulate matter samples were collected from poultry house, flourmill, textile, and food industry sites by use of liquid impinger and gravimetric samplers. Particulate matter concentrations were recorded at median concentrations of 1.56, 1.92, 4.39, and 0.7 mg/m³ in the occupied poultry house, textile, flourmill, and food indoor working environments, respectively. The highest median particulate matter concentration (27.9 mg/m³) was detected at the flourmill’s stack site. The highest median indoor concentration of culturable airborne bacteria (6.23 × 10⁵ CFU/m³) was found at the occupied poultry-house site and the lowest concentration (4.6 × 10³ CFU/m³) was found at the food industry site. The highest median indoor concentration of culturable airborne fungi (3.15 × 10⁴ CFU/m³) was found at the flourmill site whereas the lowest (1.24 × 10³ CFU/m³) was found at the textile industry site. Bacillus and Staphylococcus were the predominant Gram-positive bacteria whereas Acinetobacter and Klebsiella were the predominant Gram-negative bacteria. Escherichia coli and Salmonella were only detected in the indoor air at the poultry house site. Aspergillus flavus, Aspergillus niger, Penicillium, and yeast were the predominant fungal types at flourmill, textile, food industry, and poultry house, respectively. Workers were continuously exposed to airborne microorganisms at a median value of 10⁴ CFU/m³ in all the industries studied.     

Quantitative and qualitative study of the bacterial flora of farmed freshwater prawn (Macrobrachium rosenbergii) larvae

Quantitative and qualitative studies of the bacterial flora of farmed freshwater prawn (Macrobrachium rosenbergii) larvae in Saudi Arabia were performed, and isolates identified where possible. Physico‐chemical characteristics, bacterial counts, and the nature of the bacterial flora of larvae rearing tank water, sediment, tank wall surfaces, larval surface, supplied water, and feed were investigated. Bacterial counts ranged from 2.1 ± 1.3 × 10⁵ to 2.2 ± 0.8 × 10⁷ colony forming units (CFU) ml^?1 in tank water; 4.4 ± 0.9 × 10⁷ to 8.3 ± 1.7 ×10⁹ CFU g^?1 in tank sediment; 8.6 ± 1.0 × 10² to 9.8 ±0.7 × 10⁴ CFU cm^?2 on the tank wall surface; 1.3 ± 1.1 × 10⁴ to 7.7 ± 1.6 × 10⁶ CFU per larva surface, 7.9 ± 1.2 × 10⁵ to 5.0 ± 1.5 × 10⁷ CFU g^?1 in washed larval tissue slurries, 9.1 ± 0.7 × 10³ CFU ml^?1 in supplied water, and 2.4 ± 1.9 ×10¹⁰ CFU g^?1 in mixed feed. Fourteen bacterial genera were identified, including Chryseomonas sp., Vibrio spp., Cellulomonas sp., Aeromonas hydrophila, and Pasteurella sp. The tank water and sediment had similar bacteria to those on the prawn larvae. Chryseomonas sp., Cellulomonas sp. and Vibrio sp. were the most dominant species (prevalence >10%) in tank water; Chryseomonas sp., Pseudomonas alcaligenes and Shewanella putrefaciens in the sediment; Ps. alcaligenes and Cellulomonas sp. on the tank wall surface; Chryseomonas sp., and Cellulomonas sp. on the larval surface; and Chryseomonas sp., Vibrio vulnificus, Sh. putrefaciens and V. alginolyticus in the washed larval tissue slurries (prevalence 10%). Pseudomonas alcaligenes, Moraxella sp., Serratia liquefaciens, Gordona sp. and Burkholderia glumae were absent in larvae but identified in the culture water, tank sediment, and tank wall surface. Pseudomonas sp., Chryseomonas sp., Pasteurella sp. and V. alginolyticus were the prevalent bacteria (>12%) in supplied water. The feed contained V. alginolyticus, A. hydrophila and Cellulomonas sp. as the dominant bacteria (>13%). In the culture water and larvae samples, 83% of the feed and supplied water bacteria were identified.     

Cytotoxicity in Vero cells and cytokines analyses in Balb/c mice as safety assessments of the probiotic mixture Saccharomyces cerevisiae RC016 and Lactobacillus rhamnosus RC007 for use as a feed additive

The objective was to carry out cytotoxicity assays in Vero cells and cytokines analyses in Balb/c mice as safety assessments to evaluate the probiotic mixture (M) Saccharomyces cerevisiae RC016 (Sc) and Lactobacillus rhamnosus RC007 (Lr) for use as feed additive. Vero cells (10⁴ cells per well) were exposed to Sc (2·08 × 10⁷, 2·08 × 10⁶; 2·08 × 10⁵ cells per ml), Lr (8·33 × 10⁷; 8·33 × 10⁶; 8·33 × 10⁵ cells per ml) and their M (1 : 1). Sc concentrations did not affect the Vero cells viability; in contrast, they were lower when exposed to Lr (P ˂ 0·0001). Vero cells showed increasing viability with M decreasing concentrations (91% viability with M2). Control BALB/c mice received only phosphate buffer saline and the others received the M. The IL-10, IL-6 and TNFα concentrations from intestinal fluid were analysed and no significant differences were observed among treatments. The same occurred with the ratio between IL-10/TNF-α. Beneficial effects of probiotics are associated with the regulation of the excessive inflammatory response; it is desirable they can modulate the cytokines production only under pathological conditions. Here, M administration to healthy mice did not induce negative side effects and expands the knowledge about beneficial effects of using probiotic microorganisms in mixture for feed additives development.