Halo Zone Research Articles

Rock Phosphate Solubilisation by Strains of Penicillium Spp. Isolated from Farm and Forest Soils of three Agro Ecological Zones of Cameroon

Rock Phosphate (RP) is theoretically the cheapest fertilizer and most abundant, but its direct application in the soils is not always agronomically effective due to its low phosphorus (P) availability. An environmentally friendly and economically alternative to traditional processing of these deposits is the use of Phosphate Solubilising Microorganisms (PSM). Three Penicillium strains were screened for their aptitude in solubilising sparingly soluble phosphates as well as phosphate rock on plates and in broth supplemented with the different phosphate types. The three strains show the ability in solubilising at least two from the sparingly soluble phosphates types, with the solubilisation index (SI) varying from 1.44 to 4.74. All the strains were able to show halo zone on plates supplemented with Malian and Algerian rock phosphates with solubilisation index ranging from 1.2 to 1.8. No halo zone was detected on plates with Moroccan rock phosphate as sole phosphate source. However, significant amount of phosphorus were mobilised by all strains in broth with the different rock phosphates, including the Moroccan rock phosphate. The amount of solved P varied from 257.46 mgP.L-1 (Penicillium sp. 43) to 326.65 mgP.L-1 (Penicillium sp. 109) for Malian rock phosphate, from 152.61 mgP.L-1 (Penicillium sp. 43) to 337.56 mgP.L-1 (Penicillium sp. 27) for Algerian rock phosphate, and from 283.79 mgP.L-1 (Penicillium sp. 43) to 347.16 mgP.L-1 (Penicillium sp. 109) for Moroccan rock phosphate. The rock phosphate solubilisation was associated with pH media drop and both parameters were strongly correlated.

Rock-Phosphate Solubilising Bacteria and their Effect on Soybean (Glycine max) Growth under Pot Grown Conditions

The modern agriculture is dependent on phosphorus (P) derived from phosphate rock. However, the direct application of low-grade rock phosphate as a P source in soils need an addition of inoculums of phosphate solubilising microorganisms to improve the rock phosphate efficiency as a phosphorus source. Phosphate solubilising bacteria (PSB) were screened for their phosphate solubilising ability on plates and in liquid cultures supplemented with Malian, Moroccan or Mexican rock phosphates. They were subsequently tested on soybean grown in pots filled with non sterile soil amended with Moroccan rock phosphate for their aptitude in promoting soybean growth. The activity of the different strains on plates indicates Panthoea sp. and Bacillus sp. as the most efficient strains able to show halo zone on plates supplemented all different rock phosphates, with a solubilisation index (SI) of 3.65, 4.10 and 5.42 (Panthoea sp.) and 2.93, 3.13 and 2.13 (Bacillus sp.) respectively for Malian, Moroccan and Mexican rock phosphates. Panthoea sp. remains the strains showing the highest concentration of the solved P with all rock phosphates: 1038.25, 996.67 and 1207.87 µgP/g for Malian, Moroccan and Mexican rock phosphates respectively. It is followed by Klebsiella sp. (862.57, 615.19 and 426.29 µgP/g respectively) and Bacillus sp. (810.86, 270.92 and 180.95 µgP/g). In general, Panthoea sp. and Bacillus sp. better contribute to the soybean growth with the effect of 35% and 34% respectively compare to non inoculated control supplied with non soluble Moroccan rock phosphate. The activity of Klebsiella sp. (13%) that is low in general seems to be stimulated when associated with the two other strains (33%). This suggests that the use of rock phosphate combined with the co-inoculation with those strains would ensure soybean production in economically profitable and environmentally friendly conditions.

Fabrication and Characterization of Chitosan/Cellulose-ZnO Nanocomposites for Bactericidal Applications

ABSTRACTThe present work focuses on the fabrication of environmental friendly ZnO nanocrystals and chitosan/cellulose films hosting ZnO nanoparticles (NPs) as an attempt to produce nanocomposites with enhanced bactericidal capacity. The solution casting method was used to fabricate the chitosan/cellulose blend films. Highly monodisperse ZnO nanoparticles were synthesized using Zinc acetate and Triethylene glycol (TEG) via a modified Polyol route. ZnO crystal size was controlled by the heterogeneous nucleation approach. Optical properties of ZnO nanoparticles were studied by UV–vis spectroscopy and Photoluminescence Spectroscopy (PL) techniques. The nanoparticles’ size and morphology were determined by Transmission Electron Microscopy (TEM) and X-ray diffraction (XRD), respectively. Obtained results confirmed the effectiveness of the size-controlled synthesis employed. The chitosan/cellulose/ZnO nanocomposites were characterized by Fourier Transform – Infrared spectroscopy (FTIR) and X-ray diffraction (XRD) methods. The mechanical properties of produced bare and ZnO-bearing composites were determined from stress-strain tests. The Standard Plate Count and the Halo Zone methods were used to evaluate the bactericidal properties of the ZnO nanoparticles, chitosan/cellulose blend films and chitosan/cellulose/ZnO nanocomposites againstEscherichia coli(ATCC 35218).

Enzymatic activity profile of a Brazilian culture collection of Candida albicans isolated from diabetics and non‐diabetics with oral candidiasis

The secretion of hydrolytic enzymes is a fundamental virulence factor of Candida albicans to develop disease. The objective of this study was to characterise the virulence of 148 clinical isolates of C. albicans from oral candidiasis by assessing the expression of phospholipase (PL) and secreted aspartyl proteinase (SAP). Isolates were obtained from healthy subjects (HS) and diabetics (DOC) and non-diabetics with oral candidiasis (NDOC). An aliquot (5μl) of each cell suspension was inoculated on PL and SAP agar plates and incubated. Enzymes secretion was detected by the formation of an opaque halo around the colonies and enzymatic activity (PZ) was determined by the ratio between colony diameter and colony diameter plus the halo zone. Statistical comparisons were made by a one-way anova followed by Tukey's post hoc test (α=0.05). The clinical sources of C. albicans had significant effect (P<0.001) on the PZ values of both enzymes. For PL, clinical isolates from NDOC and DOC had highest enzymatic activity than those from HS (P<0.05), with no significant differences between them (P=0.506). For SAP, C. albicans from NDOC showed the lower enzymatic activity (P<0.001). There were no significant differences between isolates from HS and DOC (P=0.7051). C. albicans isolates from NDOC and DOC patients showed an increased production of PL.

Partial Optimization of Endo-1, 4-Β-Xylanase Production by Aureobasidium pullulans Using Agro-Industrial Residues.

Objective(s) : Although bacteria and molds are the pioneering microorganisms for production of many enzymes, yet yeasts provide safe and reliable sources of enzymes with applications in food and feed. Single xylanase producer yeast was isolated from plant residues based on formation of transparent halo zones on xylan agar plates. The isolate showed much greater endo-1, 4-β-xylanase activity of 2.73 IU/ml after optimization of the initial extrinsic conditions. It was shown that the strain was also able to produce β-xylosidase (0.179 IU/ml) and α-arabinofuranosidase (0.063 IU/ml). Identification of the isolate was carried out and the endo-1, 4-β-xylanaseproduction by feeding the yeast cells on agro-industrial residues was optimized using one factor at a time approach. The enzyme producer strain was identified as Aureobasidiumpullulans. Based on the optimization approach, an incubation time of 48 hr at 27°C, inoculum size of 2% (v/v), initial pH value of 4 and agitation rate of 90 rpm were found to be the optimal conditions for achieving maximum yield of the enzyme. Xylan, containing agricultural residues, was evaluated as low-cost alternative carbon source for production of xylanolytic enzymes. The production of xylanase enzyme in media containing wheat bran as the sole carbon source was very similar to that of the medium containing pure beechwoodxylan. This finding indicates the feasibility of growing of A. pullulans strain SN090 on wheat bran as an alternate economical substrate in order for reducing the costs of enzyme production and using this fortified agro-industrial byproduct in formulation of animal feed.

Synthesis, characterization and evaluation of bioactivity and antibacterial activity of quinary glass system (SiO2–CaO–P2O5–MgO–ZnO): In vitro study

Bioactive glasses in the systems SiO2–CaO–P2O5–MgO (BGZn0) and SiO2–CaO–P2O5–MgO–ZnO (BGZn5), were prepared by sol–gel method and then characterized. Surface reactivity was studied in simulated body fluid (SBF) to determine the effect of zinc (Zn) addition as a trace element. The effect of Zn addition to the glass matrix on the formation of apatite layer on the glass surface was investigated through X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT–IR) and scanning electron microscopy (SEM). Also, inductively coupled plasma–optical emission spectroscopy (ICP–sOES) was used to determine the concentrations of released ions in SBF solution after different time intervals in SBF solution. The antibacterial activity of Zn containing glass against Pseudomonas aeruginosa was measured by the halo zone test. The presence of Zn in glass composition improved chemical durability, slowed down the formation rate of Ca–P layer and decreased the size of crystalline apatite particles. Zn containing glass exhibited an excellent antibacterial activity against P. aeruginosa which could demonstrate its ability to treat bone infection.

English

Many phosphate solubilizing fungi (PSF) are found in soil and their introduction in the rhizosphere of crops not only increases the availability of phosphorus from insoluble sources of phosphate but also increases the efficiency of phosphate fertilizers such as superphosphate and rock phosphate. Studies on 2 strains belonging to Aspergillus niger, and 1 strain each of Aspergillus flavus, Penicillium aurantiogriseum and Penicillium claviformis with special reference to phosphate solubilization were performed in this communication. All the fungal strains showed halo zone around them on Pikovskaya plates. Quantitative estimation of 3 indigenous strains along with the other 2 non-indigenous strains taken as reference strains was done in two nutrient broths namely: Czapek’s Dox and Pikovskaya containing dicalcium phosphate (DCP) as insoluble source of phosphorus. Out of the two media selected for the study, Pikovskaya broth supported better solubilization. The insoluble phosphates DCP, TCP (tricalcium phosphate) and hypt (hydroxyapatite) were tested, DCP was least solubilized by all strains but there was no significant difference in solubilization of TCP and hydroxyapatite. There was significant negative correlation between pH and phosphate solubilized in all forms of insoluble phosphates. Types of nitrogen sources and metal ions were also screened during the study. (Na4)2SO4 supported more solubilization than NaNO3, whereas phosphate solubilizing activity decreased (except in A. niger ATCC 282) in presence of Mn+2 and Fe+3 but presence of Al+3 did not have statistically significant effect on solubilization. Though various species of Penicillium have been reported for phosphate solubilization in literature but to the best of our knowledge this is first report of phosphate solubilization by P. claviformis. Key words: Aspergillus, Penicillium, phosphate solubilisation, dicalcium phosphate, tricalcium phosphate, hydroxyapatite.

Identification of the haemolytic activity of Malassezia species

Malassezia species are part of the normal skin flora and are associated with a number of human and animal skin diseases. However, the mechanisms that mediate infection and host-fungal interactions are poorly understood. The haemolytic activity of several microorganisms is considered a factor that contributes to pathogenicity of the organism to humans and animals. This virulence factor was previously identified in several pathogenic fungi that cause systemic mycoses, such as Aspergillus and Candida. In this study, the haemolytic activity of six major Malassezia species, including M. furfur, M. globosa, M. pachydermatis, M. restricta, M. slooffiae and M. sympodialis, was investigated. The haemolytic activity of these species was tested on tryptone soya agar with 5% sheep blood. All the examined Malassezia species produced a halo zone of complete haemolysis. A quantitative analysis of the haemolytic activity was performed by incubating sheep erythrocytes with the extraction from culture of each Malassezia species. Interestingly, M. globosa and M. restricta showed significantly high haemolytic activity compared with the other Malassezia species. In addition, M. globosa also exhibited stable haemolytic activity after treatment at 100°C and in the presence of some proteases, indicating that this haemolytic factor is different from those of other fungi.

Isolation and characterization of beneficial indigenous endophytic bacteria for plant growth promoting activity in Molelwane Farm, Mafikeng, South Africa

Plant-associated bacteria that live inside plant tissues without causing any damage to plants are defined as endophytic bacteria. The present study was carried out to analyze the phenotypic and genotypic diversity of endophytic bacteria associated with Amaranthus hybridus, Solanum lycopersicum and Cucurbita maxima . A total of 50 bacteria were isolated from the roots of the plants. All the isolates were screened for morphological features (Gram reaction, pigmentation, odour, colour, motility and sharp). Isolates exhibiting difference in morphological features were selected for molecular identification. Eight isolates that exhibited differences in phenotypic aspect were subjected to partial 16S-rDNA gene sequencing using polymerase chain reaction (PCR) for phylogenetic analysis. Sequence analysis using Clustal-X version 1.83 software identified the following isolated bacteria: Stenotrophomonas maltophilia KC010525, Pseudomonas putida KC010526, P. putida KC010527, P. putida KC010528, S. maltophilia KC010529, Achromobacter xylosoxidans KC010530, A. xylosoxidans KC010531 and Achromobacte sp. KC010532. Further evaluation of the bacterial isolates for phosphate solubilization capacity, indole acetic acid (IAA), hydrogen cyanide (HCN) and ammonium gas production, showed all eight bacterial isolates were able to produce IAA (0.32-2.42 mg/mgl -1 ). However, seven isolates excluding S. maltophilia KC010525 showed ability to produce ammonium. HCN was observed in six isolates: A. xylosoxidans KC010530, A. xylosoxidans KC010531, A. KC010532, P. putida KC010526, P. putida KC010527, and P. putida KC010528. When determining the phosphate solubilizing capacity, it was observed that seven solubilized insoluble phosphate in Pikovskya’s agar plates produced halo zones (1 to 4 mm). Seven tested bacteria were active against Fusarium oxysporum. Therefore, the results indicate that the bacteria isolates may be used as a promising microbial inoculant for plant growth and productivity. Keywords : Plant growth promoting rhizobacteria (PGPR), 16S-rDNA sequencing, HCN production, indole acetic acid (IAA), phosphate solubilization, antifungal activity. African Journal of Biotechnology Vol. 12(26), pp. 4105-4114

Isolation and identification of phytate-degrading rhizobacteria with activity of improving growth of poplar and Masson pine

A number of soil microorganisms can convert insoluble forms of phosphorus (P) to an accessible form to increase plant yields. Phytate is such a large kind of insoluble organic phosphorus that plants cannot absorb directly in soil, so the objectives of this study were to isolate, screen phytate-degrading rhizobacteria (PDRB), and to select potential microbial inocula that could increase the P uptake by plants. In this study, a total of 24 soil samples were collected from natural habitats of eight poplar and pine planting areas from the eastern to southern China. 17 PDRB strains were preliminarily screened from the rhizosphere soil of poplars and pines by the visible decolorization in the phytate selective medium. The highest ratio of the total diameter (colony + halo zone) to the colony diameter of the isolates was JZ-GX1, 3.85. Afterward, 17 PDRB strains were further determined for their abilities to degrade sodium phytate based on the amount of liberated inorganic P in liquid phytate specific medium. The results showed that the phytase ability of the three highest PDRB strains: JZ-GX1, JZ-DZ1 and JZ-ZJ1 were up to 2.58, 2.36 and 2.24 U/mL, respectively, much better than most of the bacteria reported in previous studies. In the soil-plant experiment, compared to CK, the best three strains of PDRB all could significantly promote growth of poplar and Masson pine under container growing. The three efficient PDRB strains were identified as follow: JZ-GX1, Rahnella aquatilis, both JZ-DZ1 and JZ-ZJ1 being autofluorescent, Pseudomonas fluorescens, by 16S rDNA gene sequencing technology, Biolog Identification System and biological characterization. The present study suggests that the three screened PDRB strains would have great potential application as biological fertilizers in the future.

Fabrication and characterization of antibacterial PVDF hollow fibre membrane by doping Ag-loaded zeolites

This work describes the fabrication and characterization of antibacterial poly(vinylidene fluoride) (PVDF) hollow fibre membranes via phase inversion using dry-jet wet-spinning technique. Ag-loaded NaY zeolites were particularly synthesized and then doped with PVDF spinning solution to produce a novel antibacterial hollow fibre with enhanced antibacterial efficiency. The morphologies, crystal structures, and Ag+ loading of synthesized Ag-loaded NaY zeolites were analyzed by field emission scanning electron microscope (FE-SEM), X-ray diffraction (XRD) and inductively coupled plasma-atomic emission spectrometry (ICP-AES), respectively. The antibacterial efficiencies of the prepared hollow fibre membranes against Escherichia coli (E. coli) were evaluated by halo zone test, E. coli suspension filtration test and adhesion test. The surface morphologies, pure water flux, hydrophilicity, tensile strength, through-pore size, resistance to BSA of antibacterial PVDF hollow fibre membrane were also investigated. All results demonstrated that PVDF hollow fibre fabricated by doping Ag-loaded zeolites showed excellent antibacterial property, high permeability and mechanical strength, indicating the promising application as the antibacterial ultra-filtration membranes.

Production of extracellular enzymes in the entomopathogenic fungus Verticillium lecanii

This study investigates the mechanisms as well as strategies for purification and characterization of potential enzymes involved in pathogenesis of entomopathogenic fungi. The test strain of Verticillium lecanii that was screened, during the present investigation, proved to be an efficient producer of protein and polysaccharide degrading enzymes (amylase, protease, and lipase), hence indicating versatility in biochemical mechanisms. Halo zones produced colony growth of V. lecanii on agar confirmed activity of protease, amylase and lipase enzyme by the V. lecanii isolate. Enzymatic Index (EI) observed were: Protease – 2.195, Amylase- 2.196, Lipase- 2.147. Spectrophotometric analysis of enzymatic activity of V.lecanii at five different pH – 3, 5, 7, 9, 11 revealed that highest proteolytic activity of the V. lecanii isolate was reported at pH 7 and 9 whereas proteolytic activity was minimum at acidic pH 3. Maximum amylolytic activity of V. lecanii on the 7th day of inoculation was at pH 3 i.e. in an acidic environment in contrast to neutral pH 7. Maximum lipolytic activity of V. lecanii was found at pH 7. Since enzyme production in entomopathogenic fungi is specific and forms an important criterion for successful development as well as improvement of mycoinsecticides, hence a significant conclusion from the present analysis is the degree of variation in secretion of enzymes in test strain of Verticillium lecanii.

Reactive wetting of Sn–2.5Ag–0.5Cu solder on copper and silver coated copper substrates

In the present work, wetting characteristics and morphology of intermetallic compounds (IMCs) formed between Sn–2.5Ag–0.5Cu lead-free solder on copper (Cu) and silver (Ag) coated copper substrates were compared. It was found that, Ag coated Cu substrate improved the wettability of solder alloy. The average values of contact angles of solder alloy solidified on Ag coated Cu substrate were reduced to about 50 % as compared to contact angles obtained on Cu substrates. Flow restrictivity for spreading of solder on Ag coated Cu was found to be lower as compared to Cu substrate. The spreading of solder alloy on Ag coated Cu exhibited halo zone. Coarse needle shaped Cu6Sn5 IMCs were observed at the solder/Cu substrate interface whereas at the solder/Ag coated Cu interface Cu6Sn5 IMCs showed scallop morphology. The formation of Cu3Sn IMC was observed for the spreading of solder alloy on both substrates. The solder/Ag coated Cu substrate interface exhibited more particulates of Ag3Sn precipitates as compared to solder/Cu substrate interface. The improved wettability of solder alloy on Ag coated Cu substrate is due to the formation of scallop IMCs at the interface.

Molecular identification of a novel inulinolytic fungus isolated from and grown on tubers of Helianthus tuberosus and statistical screening of medium components

Of 24 fungi belonging to more than five genera isolated from tubers of rotten Helianthus tuberosus, 11-inulinolytic active isolates were able to develop halo zones around their fungal colonies, indicating inulinase activity. Alternaria, Aspergillus, Fusarium, Pencillium and Trichoderma were the most common inulinolytic genera, representing more than 90 % of the total positive inulinolytic fungi. Aspergillus tamarii and Pencillium citrinum quantitatively recorded better growth (5.5 and 4.7 mg ml(-1)) and inulinase production (21.53 and 20.15 U ml(-1)) in submerged culture. The enzyme preparation showed also invertase activity. Aspergillus tamarii, as the most potent producer of inulinase, was identified using the Inter Transcribed Spacer marker. The sequence comparisons showed that our molecularly identified strain (GU295949) is related more closely to A. tamarii strains of the gene bank. Statistical screening using the fractional factorial Plackett-Burman design with 12 run was applied for screening ten variables, the low levels of pH (4.8), inoculum size (10(3) spore g(-1)), NH(4)NO(3) (1.0 mg g(-1)) and MgSO(4) (0.12 mg g(-1)), were the most significant variables on A. tamarii inulinase production. The high inulinase/invertase ratio (1.841-4.293) classified the enzyme preparation as inulinases, which can be used efficiently in production of fructose syrup from tubers of H. tuberosus.

Focal High Cell Density Generates a Gradient of Patterns in Self-Organizing Vascular Mesenchymal Cells

In embryogenesis, structural patterns, such as vascular branching, may form via a reaction-diffusion mechanism in which activator and inhibitor morphogens guide cells into periodic aggregates. We previously found that vascular mesenchymal cells (VMCs) spontaneously aggregate into nodular structures and that morphogen pairs regulate the aggregation into patterns of spots and stripes. To test the effect of a focal change in activator morphogen on VMC pattern formation, we created a focal zone of high cell density by plating a second VMC layer within a cloning ring over a confluent monolayer. After 24 h, the ring was removed and pattern formation monitored by phase-contrast microscopy. At days 2–8, the patterns progressed from uniform distributions to swirl, labyrinthine and spot patterns. Within the focal high-density zone (HDZ) and a narrow halo zone, cells aggregated into spot patterns, whilst in the outermost zone of the plate, cells formed a labyrinthine pattern. The area occupied by aggregates was significantly greater in the outermost zone than in the HDZ or halo. The rate of pattern progression within the HDZ increased as a function of its plating density. Thus, focal differences in cell density may drive pattern formation gradients in tissue architecture, such as vascular branching.

Integrating a scalable and effcient semi-Lagrangian multi-tracer transport scheme in HOMME

With the inclusion of a scalable spectral-element-based version of the High-Order Method Modeling Environment (HOMME) in NCAR's Community Atmosphere Model (CAM) and the need for on the order of 100 prognostic tracers in next generation climate modeling efforts, there is an urgent need for a scalable, monotonic, and conservative multitracer advection scheme in HOMME. To address this, we integrate a Conservative Semi-LAgrangian Multi-tracer transport scheme (CSLAM) into HOMME. The algorithm is specifically re-designed for multiple processors and the cubed-sphere grid with the element-wise domain decomposition as employed by HOMME. Based on these spectral elements, we create a finite volume mesh and add a halo zone of cells since a higher-order CSLAM needs a patch of tracer values for a reconstruction that extend the element. Therefore, we extend HOMME to also be capable of communicating cell center data. Our data structures and design allow us to provide a numerical scheme that still conserves mass locally to machine precision and is also monotone on multiple processor systems, but reduces the communication of tracer values by one for each time step. This is based on the idea that we calculate the reconstruction coeffcients redundantly, in a cell of an element and in the corresponding cell in the halo zone. Our algorithm of CSLAM in HOMME is the first one for multiple processor systems and multi-tracers that is highly scalable and effcient.

X射线相衬成像中靶丸球壳界面晕区暗区现象

X射线相衬成像中靶丸球壳界面晕区暗区现象

Effect of Signal Peptides on the Secretion of β-Cyclodextrin Glucanotransferase in Lactococcus lactis NZ9000

Cyclodextrin glucanotransferase (CGTase) is an extracellular enzyme which catalyzes the formation of cyclodextrin from starch. The production of CGTase using lactic acid bacterium is an attractive alternative and safer strategy to produce CGTase. In this study, we report the construction of genetically modified Lactococcus lactis strains harboring plasmids that secrete the Bacillus sp. G1 β-CGTase, with the aid of the signal peptides (SPs) SPK1, USP45 and native SP (NSP). Three constructed vectors, pNZ:NSP:CGT, pNZ:USP:CGT and pNZ:SPK1:CGT, were developed in this study. Each vector harbored a different SP fused to the CGTase. The formation of halo zones on starch plates indicated the production and secretion of β-CGTase by the recombinants. The expression of this enzyme is shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis. A band size of ∼75 kDa corresponding to β-CGTase is identified in the intracellular and the extracellular environments of the host after medium modification. The replacement of glucose by starch in the medium was shown to induce β-CGTase production in L. lactis. Although β-CGTase production is comparatively low in NZ:SPK1:CGT, the SP SPK1 was shown to have higher secretion efficiency compared to the other SPs used in this study.

Measuring Bacterial Load and Immune Responses in Mice Infected with &lt;em&gt;Listeria monocytogenes&lt;/em&gt;

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8α+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6 . There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to β-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with &lt;em&gt;Listeria monocytogenes&lt;/em&gt;

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8α+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6. Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6 . There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design. We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to β-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.