In vitro efficacy of nisin Z against Candida albicans adhesion and transition following contact with normal human gingival cells

Aim:  To investigate the nisin Z innocuity using normal human gingival fibroblast and epithelial cell cultures, and its synergistic effect with these gingival cells against Candida albicans adhesion and transition from blastospore to hyphal form.
Methods and Results:  Cells were cultured to 80% confluence and infected with C. albicans in the absence or presence of various concentrations of nisin Z. Our results indicate that only high concentrations of nisin Z promoted gingival cell detachment and differentiation. Determination of the LD₅₀ showed that the fibroblasts were able to tolerate up to 80  μ g ml⁻¹ for 24 h, dropping thereafter to 62  μ g ml⁻¹ after 72 h of contact, compared to 160  μ g ml⁻¹ after 24 h, and 80  μ g ml⁻¹ after 72 h recorded by the gingival epithelial cells which displayed a greater resistance to nisin Z. The use of nisin Z even at low concentration (25  μ g ml⁻¹) at appropriate concentrations with gingival cells significantly reduced C. albicans adhesion to gingival monolayer cultures and inhibited the yeast's transition.
Conclusion:  These findings show that when used at non-toxic levels for human cells, nisin Z can be effective against C. albicans adhesion and transition and may synergistically interact with gingival cells for an efficient resistance against C. albicans .
Significance and Impact of the Study:  This study suggests the potential usefulness of nisin Z as an antifungal agent, when used in an appropriate range.     

Development and characterization of a lux-modified 2,4-dichlorophenol-degrading Burkholderia sp. RASC

lux -marked biosensors for assessing the toxicity and bioremediation potential of polluted environments may complement traditional chemical techniques. lux CDABE genes were introduced into the chromosome of the 2,4-dichlorophenol (2,4-DCP)-mineralizing bacterium, Burkholderia sp. RASC c2, by biparental mating using the Tn 4431 system. Experiments revealed that light output was constitutive and related to cell biomass concentration during exponential growth. The transposon insertion was stable and did not interrupt 2,4-DCP-degradative genes, and expression of lux CDABE did not constitute a metabolic burden to the cell. A bioluminescence response was detectable at sublethal 2,4-DCP concentrations: at < 10.26 μg ml⁻¹, bioluminescence was stimulated (e.g. 218% of control), but at concentrations > 60 μg ml⁻¹ it declined to < 1%. Investigating the effect of [14C]-2,4-DCP concentration on the evolution of ¹⁴CO₂ revealed that, for initial concentrations of 2.5–25 μg ml⁻¹, ≈55% of the added ¹⁴C was mineralized after 24 h compared with < 1% at 50 and 100 μg ml⁻¹. Inhibition of 2,4-DCP mineralization between 25 and 50 μg ml⁻¹ corresponded well to the EC₅₀ value (33.83 μg ml⁻¹) obtained from bioluminescence inhibition studies. lux -marked RASC c2 may therefore be used as a functionally (i.e. 2,4-DCP degrader) and environmentally relevant biosensor of toxicity and biodegradation inhibition.     

Response of Listeria monocytogenes to liquid smoke

Aims:  To investigate the effect of liquid smoke on growth, survival, proteomic pattern and haemolytic potential of Listeria monocytogenes.
Methods and Results:  Growth and survival curves were recorded in brain–heart infusion broth supplemented with three concentrations of liquid smoke. L. monocytogenes growth was inhibited in the presence of 15 μg ml⁻¹ phenol while a rapid decrease in cell viability occurred in the presence of 30 μg ml⁻¹ phenol. The proteome of L. monocytogenes cytosoluble proteins was slightly modified after 2-h incubation with 30 μg ml⁻¹ phenol but no protein already characterized in response to other known stresses was induced, except the protease ClpP. Liquid smoke inhibited the haemolytic potential without affecting hly gene expression, showing a potential inhibition of protein activity or stability.
Conclusions:  The presence of liquid smoke in a rich medium strongly affected growth and survival of L. monocytogenes . Brief smoke stress affected the metabolic pathways and inhibited the haemolytic activity of L. monocytogenes .
Significance and Impact of Study:  This study is a first step in the investigation of the influence of a smoked product on L. monocytogenes strains.     

Inhibitory effects of sucrose fatty acid esters on survival of Yersinia enterocolitica on eggshell surface and use of blue lake as indicator of bacterial penetration into eggs

Aims:  To determine the effectiveness of sucrose monolaurate (SML) and sucrose monocaprate (SMC), alone and in combination with ethylenediaminetetraacetic acid (EDTA), propionic acid (PA) or citric acid (CA) in reducing mesophilic aerobic bacteria (MAB) and Yersinia enterocolitica O:9 populations on eggshells and their damage potential on the microstructure of shell cuticle.
Methods and Results:  Uninoculated eggs and eggs submerged in a solution of Y. enterocolitica were immersed in solutions of the various treatments. MAB and Y. enterocolitica counts on the surface of the eggs were carried out before and after treatment. MAB counts decreased less than 2 logs on uninoculated eggshells irrespective of treatment and reductions of 3·2 and 3·0 logs of Y. enterocolitica were obtained with 1000 μg ml⁻¹ SML plus 0·1% CA or 1000 μg ml⁻¹ SML plus 600 μg ml⁻¹ EDTA solutions, respectively. Y. enterocolitica 2/O:9 was recovered from natural microflora. Use of blue lake staining revealed minimal damage to the shells from the washing treatments.
Conclusions:  SML and SMC at 1000 μg ml⁻¹ combined with CA or EDTA could be effective in reducing Y. enterocolitica on eggshells with a minimal risk of later bacterial recontamination.
Significance and Impact of the Study:  Eggs are a recognized vehicle for transmission of Y enterocolitica although a prevalence of only 2·7% was detected in this study. Washing eggs in solutions containing SML or SMC could eliminate Y. enterocolitica contamination of egg shells.     

Trichophyton species susceptibility to green and red propolis from Brazil

Aims:  The in vitro antifungal activity of Brazilian green and red propolis was tested against different species of Trichophyton .
Methods and Results:  The antifungal activity of the Brazilian aqueous and alcoholic extracts of the green propolis and the alcoholic extract of red propolis was observed against Trichophyton rubrum , Trichophyton tonsurans and Trichophyton mentagrohytes samples, using as controls itraconazole and terbinafine. The minimal inhibitory concentration was determined following the microdilution method indicated by the 'Clinical and Laboratory Standards Institute'. The minimal fungicide concentration was determined by the absence of growth in liquid sabouraud culture medium. The data obtained showed that the green propolis alcoholic extract's antifungal activity was from 64 to 1024 μg ml⁻¹, whereas the red propolis alcoholic extract was from 8 to 1024 μg ml⁻¹.
Conclusions:  The antifungal activity of the red propolis alcoholic extract was more efficient than the green propolis alcoholic extract for all three species studied. The T. rubrum samples were shown to be more sensitive to the antifungal activity of the alcoholic extracts of the propolis.
Significance and Impact of the Study:  The antifungal potential of the alcoholic extracts of green and red propolis demonstrated suggest an applicable potential as an alternative treatment for dermatophytosis caused by these species.     

Antimicrobial activity of essential oils and structurally related synthetic food additives towards Clostridium perfringens

Aims:  To assess the potential of essential oils and structurally related synthetic food additives in inhibiting the growth of Clostridium perfringens for the control of necrotic enteritis in chickens.
Methods and Results:  The antimicrobial activity of essential oils/compounds was measured by determining the inhibition of bacterial growth. Thirty-three of 66 oils/compounds exhibited ≥80% inhibition. Seven with the highest potency were further studied. The oils/compounds had MIC₉₅ values between 167 and 425  μ g ml⁻¹. Most of them were tolerant to low pH (2·0) and exhibited minor or no inhibition of Lactobacillus isolates from the chicken intestine. When mixed with chicken ileal digesta, the oils/compounds retained their efficacy against C . perfringens , but had little effect on the total number of lactobacilli and anaerobic bacteria in the digesta.
Conclusions:  Some essential oils/compounds demonstrated good potential in controlling C . perfringens .
Significance and Impact of the Study:  This study has identified candidates of essential oils/compounds for in vivo studies for the control of necrotic enteritis in chickens.     

Investigation of the anti-fungal activity of coptisine on Candida albicans growth by microcalorimetry combined with principal component analysis

Aims:  This study investigated the anti-fungal activity of coptisine on Candida albicans growth.
Methods and Results:  The metabolic power-time curves of Candida albicans growth at 37°C affected by coptisine were measured by microcalorimetry using an LKB-2277 Bioactivity Monitor with stop-flow mode. Then, the diameter of inhibitory zones in the agar layer was observed using agar cup method, and the minimal inhibitory concentration (MIC) of coptisine on Candida albicans growth was determined by serial dilution method. From the principal component analysis on nine quantitative parameters obtained from the power-time curves, we could easily evaluate the anti-fungal activity of coptisine by analysing the change of values of the main two parameters, growth rate constant k and maximum power output in the log phase P _{m, log}. The results showed that coptisine had strong anti-fungal activity: at a low concentration (45  μ g ml⁻¹) began to inhibit the growth of Candida albicans and at a high concentration (500  μ g ml⁻¹) completely inhibited Candida albicans growth. Coptisine gave big inhibitory zones with diameters between 11 and 43 mm within test range, and the MIC of it was 1000  μ g ml⁻¹.
Conclusions:  Coptisine had strong anti-fungal activity on Candida albicans growth. The method of microcalorimetry applied for the assay of anti-fungal activity of coptisine was quantitative, sensitive and simple.
Significance and Impact of the Study:  This work will provide useful information for the development of chemical biology policy in the use of anti-microbials in food and drug production.     

Acrebol, a novel toxic peptaibol produced by an Acremonium exuviarum indoor isolate

Aims:  To identify a toxin and its producer isolated from woody material in a building where the occupants experienced serious ill health symptoms.
Methods and Results:  Hyphal extracts of an indoor fungus, identified as the cycloheximide-tolerant species Acremonium exuviarum , inhibited motility of boar spermatozoa (EC₅₀ 5 ± 2 μg of crude solids ml⁻¹) and caused cytolysis of murine neuroblastoma cells (MNA) and feline fetal lung cells (FL). The responsible substances were purified and identified as two structurally similar, heat-stable, novel, toxic peptaibols, 1726 Da and 1740 Da, respectively, with amino acid sequences of Acetyl-Phe-Iva/Val-Gln-Aib-Ile-Thr-Leu-Aib-Pro-Aib-Gln-Pro-Aib-(X-X-X)-SerOH and Acetyl-Phe-Iva/Val-Gln-Aib-Ile-Thr-Leu-Val-Pro-Aib-Gln-Pro-Aib-(X-X-X)-SerOH. Purified acrebol inhibited motility of boar sperm, depleted ATP half-content in 1 day (EC₅₀ of 0·1 μg ml⁻¹, 60 nmol l⁻¹) depolarised the mitochondria after 2 days, but did not affect the cellular content in NADH. This indicates mitochondrial toxicity. Plate-grown biomass of A. exuviarum BMB4 contained 0·1–1% (w/w) of acrebol, depending on the culture medium.
Conclusions:  Acrebol paralysed the energy generation of mammalian cells suggesting that mitochondria were its target of action.
Significance and Impact of the Study:  Acremonium exuviarum, as an indoor fungus, is potentially hazardous to health because of the toxic peptaibols that it produces.     

Optimization and biochemical characterization of a bacteriocin from a newly isolated Bacillus subtilis strain 14B for biocontrol of Agrobacterium spp. strains

Aims:  The identification of a new compound active against Agrobacterium tumefaciens .
Methods and Results:  The culture conditions of a newly isolated Bacillus subtilis strain, designed 14B, were optimized, as a first step, to produce its bacteriocin (termed Bac 14B) for the biocontrol of Agrobacterium spp., the causal agents of the crown gall disease. Bac 14B was then partially purified and biochemically characterized. Bacillus subtilis 14B was observed to produce an antibacterial compound having a protinaceous nature. As estimated by sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE), the semi-purified bacteriocin substance was found to be a monomeric protein with a molecular weight of 21 kDa. While the latter's antimicrobial activity was completely stable during exposure to a temperature range of up to 100°C for 2 h, its initial activity was totally lost at 121°C for 20 min. The maximum bacteriocin production (4096 AU ml⁻¹) was recorded after 96 h-incubation in an optimized Luria Bertani medium supplemented with 10 g l⁻¹ glucose, 15 g l⁻¹ K₂HPO₄ and 5 g l⁻¹ MgSO₄ 7H₂O at 30°C in a shaking flask culture. Interestingly, the B. subtilis 14B culture supernatant that contained the bacteriocin under study was proved efficient in reducing both the percentage of galled plants and the number of galls in tomato.
Conclusion:  The findings revealed that B. subtilis 14B and its bacteriocin are efficient in reducing the percentage of infections in plants caused by Ag. tumefaciens .
Significance and Impact of the Study:  The results could be useful for the nurserymen who are particularly interested in the biocontrol of the crown gall disease.     

Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine

Aims:  Brettanomyces / Dekkera bruxellensis is a particularly troublesome wine spoilage yeast. This work was aimed at characterizing its behaviour in terms of growth and volatile compound production in red wine.
Methods and Results:  Sterile red wines were inoculated with 5 × 10³ viable cells ml⁻¹ of three B. bruxellensis strains and growth and volatile phenol production were followed for 1 month by means of plate counts and gas chromatography-mass spectrometry (GC-MS) respectively. Maximum population levels generally attained 10⁶–10⁷ colony forming units (CFU) ml⁻¹ and volatile phenol concentrations ranged from 500 to 4000 μg l⁻¹. Brettanomyces bruxellensis multiplication was also accompanied by the production of organic acids (from C₂ to C₁₀), short chain acid ethyl-esters and the 'mousy off-flavour' component 2-acetyl-tetrahydropyridine.
Conclusions:  Different kinds of 'Brett character' characterized by distinct metabolic and sensory profiles can arise in wine depending on the contaminating strain, wine pH and sugar content and the winemaking stage at which contamination occurs.
Significance and Impact of the Study:  We identified new chemical markers that indicate wine defects caused by B. bruxellensis. Further insight was provided into the role of some environmental conditions in promoting wine spoilage.     

A potent anti-oxidant property: fluorescent recombinant α-phycocyanin of Spirulina

Aims:  To express and product a fluorescent antioxidant holo-α-phycocyanin (PC) of Spirulina platensis ( Sp ) with His-tag (rHHPC; recombinant holo-α-phycocyaninof Spirulina platensis with His-tag) in 5-l bench scale.
Methods and Results:  A vector harbouring two cassettes was constructed: cpcA along with cpcE - cpcF in one cassette; ho1 - pcyA in the other cassette. Lyases CpcE/F of Synechocystis sp. PCC6803 ( S6 ) could catalyse the 82 site Cys in apo-α-PC of Sp linking with bilin chromophores, and rHHPC was biosynthesized in Escherichia coli BL21. The constant feeding mode was adopted, and transformant reached the biomass of rHHPC up to 0·55 g l⁻¹ broth in 5-litre bench scale. rHHPC was purified by Ni²⁺ affinity column conveniently. The absorbance and the fluorescence emission spectra of rHHPC had λ_max at 621 and 650 nm, respectively. The IC₅₀ values of rHHPC were 277·5 ± 25·8 μ g ml⁻¹ against hydroxyl radicals and 20·8 ± 2·2  μ g ml⁻¹ against peroxyl radicals.
Conclusions:  Combinational biosynthesis of rHHPC was feasible, and the constant feeding mode was adopted to produce good yields of rHHPC. Fluorescent rHHPC with several unique qualitative and quantitative features was effective on scavenging hydroxyl and peroxyl radicals.
Significance and impact of the study:  A potent antioxidant rHHPC was co-expressed, produced and characterized for nutritional and pharmacological values, which would help to develop phycobiliproteins' applications in their fluorescent and biological activities.     

Ascherxanthone B from Aschersonia luteola, a new antifungal compound active against rice blast pathogen Magnaporthe grisea

Aims:  Fungicide resistance now exists in the rice blast fungus, Magnaporthe grisea , necessitating the need for new active agents. Fungi isolated from habitats in Thailand were screened with reference to this problem.
Methods and Results:  A new, reliable in vitro screening system based on a microdilution plate format was set up using a virulent strain of M. grisea THL 16. Culture broth extracts from approximately 800 fungal strains were investigated, one of these, Aschersonia luteola BCC 8774, was found to produce an active fungicidal compound, ascherxanthone B, with an IC₉₀ value of 0·58 μg ml⁻¹ (0·95 μmol l⁻¹). An in vivo study of anti-blast efficacy of ascherxanthone B showed a positive effect in disease reduction.
Conclusions:  Previous report has shown that a species of Aschersonia produces ascherxanthone A. Research on the species, A. luteola BCC 8774, led to the discovery of related novel metabolite, ascherxanthone B with fungicidal properties.
Significance and Impact of the Study:  Current methods of rice blast control seem to fail leading to increase in crop losses. Our discovery of the anti-blast activity shown by ascherxanthone B is the first step in the development of a potentially novel fungicide.     

Antimicrobial and antistaphylococcal biofilm activity from the sea urchin Paracentrotus lividus

Aims:  Staphylococcal biofilm-associated infections are resistant to conventional antibiotics. Consequently, new agents are needed to treat them. With this aim, we focused on the effector cells (coelomocytes) of the sea urchin Paracentrotus lividus immune system.
Methods and Results:  We tested the activity of the 5-kDa peptide fraction of the cytosol from coelomocytes (5-CC) against a group of Gram-positive, Gram-negative bacteria and fungi. We determined minimal inhibitory concentrations (MICs) ranging from 253·7 to 15·8 mg ml⁻¹. We observed an inhibitory activity and antibiofilm properties of 5-CC against staphylococcal biofilms of reference strains Staphylococcus epidermidis DSM 3269 and Staphylococcus aureus ATCC 29213. The antimicrobial efficacy of 5-CC against the biofilms of clinical strain Staph. epidermidis 1457 was also tested using live/dead staining in combination with confocal laser scanning microscopy. At a sub-MIC concentration (31·7 mg ml⁻¹) of 5-CC the formation of young (6-h old) and mature (24-h old) staphylococcal biofilms was inhibited.
Conclusions:  The biological activity of 5-CC could be attributed to three peptides belonging to the sequence segment 9–41 of a beta-thymosin of P. lividus .
Significance and Impact of the Study:  The effector cells of P. lividus represent an interesting source of marine invertebrates-derived antimicrobial agents in the development of new strategies to treat staphylococcal biofilms.     

Effects of chlorophyllin on replication of poliovirus and bovine herpesvirus in vitro

Aims:  Chlorophyllin (CHLN), a synthetic derivative of chlorophyll, was assayed in the replication of poliovirus (PV-1) and bovine herpesvirus (BoHV-1) in HEp-2 cell cultures.
Methods and Results:  Virucidal activity of CHLN was evaluated and the time-of-addition assay was performed as follows: before the infection (−1 and −2 h), at the time of the infection (0 h) and after the infection (1 and 2 h). Plaque reduction assay (PRA) showed that CHLN inhibited BoHV-1 and PV-1 infection and the 50% inhibitory concentrations (IC₅₀) against BoHV-1 and PV-1 infection were 8·6 and 19·8 μg ml⁻¹, respectively. The time-of-addition study demonstrated that the CHLN was effective inhibiting viral replication in 51% and 66·5% for PV-1 and BoHV-1, respectively, at the highest concentration of 20·0 μg ml⁻¹, when added during the infection. The directed effect of CHLN on viral strains demonstrated an inhibition of 62% and 66·4% for PV-1 and BoHV-1, respectively, by PRA.
Conclusions:  These results demonstrated that CHLN could be used as an antiviral suggesting directed activity on virus particles and on virus-receptor sites to BoHV. For poliovirus, CHLN also demonstrated virucide activity, moreover, showed to inhibit early steps of the replication cycle.
Significance and Impact of the Study:  CHLN demonstrated promising selectivity index for both virus strains; therefore, it can be used for the development of an antiviral agent.     

Comparison of methods to detect resistance of Penicillium expansum to thiabendazole

Aims:  Because of the lack of a standard method, the aim of this work is to evaluate the suitability of the broth microdilution method CLSI M38-A in determining the resistance level of some Penicillium expansum isolates to thiabendazole (TBZ). The ability of the isolates to produce patulin (PAT) and citrinin (CIT) has been also assessed.
Methods and Results:  Penicillium expansum isolates (128) were assayed (apples, pears, grapes and five reference strains). It was observed that 69·4% of the strains isolated from apples and pears were resistant to TBZ. Sensitive isolates were inhibited at 0·25–0·5 μg ml⁻¹ whilst resistant isolates still grew at 512 μg ml⁻¹. PAT was produced by all P. expansum isolates. CIT was detected in 98·8% of TBZ-resistant isolates and in 89·1% of the TBZ-sensitive isolates.
Conclusions:  The preliminary screening method combined with the adaptation of the method CLSI M38-A, can be a good strategy to be used in assessing the in vitro activity of TBZ against a large number of isolates.
Significance and Impact of the Study:  The proposed methodology can be a contribution to the standardization of susceptibility tests to fungicides against P. expansum.     

Response surface methodology to optimize the nutritional parameters for enhanced production of jasmonic acid by Lasiodiplodia theobromae

Aims:  To find out the cumulative effect of the nutritional parameters and to enhance the production of jasmonic acid (JA) in static fermentation by Lasiodiplodia theobromae using response surface methodology (RSM).
Method and Results:  Malt extract, sucrose, NaNO₃ and MgSO₄.7H₂O were analysed by a 30-trial central composite design using RSM for optimizing their concentrations in the medium and the effect of their mutual interaction on JA production. Sucrose and NaNO₃ were found highly significant in influencing the JA production. Malt extract and MgSO₄.7H₂O showed an effect on the JA production in interaction with other variables. When the optimum values of the parameters obtained through RSM (19·95 g l⁻¹ malt extract, 50 g l⁻¹ sucrose, 7·5 g l⁻¹ NaNO₃ and 3·51 g l⁻¹ MgSO₄.7H₂O) were applied, 32% increase in JA production (299 mg l⁻¹) was observed in comparison with 225 mg l⁻¹ of JA produced with same media components not analysed by RSM and subsequently validated the statistical model.
Conclusions:  Increase in JA production was achieved by optimizing the nutritional parameters.
Significance and Impact of the Study:  This is the first report of using RSM for optimizing a medium for JA production. It resulted in an increase in JA production without augmentation of costly additives.     

Heyneanol A induces apoptosis via cytochrome c release and caspase activation in human leukemic U937 cells

Heyneanol A, a tetramer of resveratrol, is isolated from the roots of Vitis amurensis by cytotoxicity based fractionation. In this study, the mechanism of apoptosis by heyneanol A was evaluated in human leukemic U937 cells. Heyneanol A (IC(50) = 6.6 microM at 24 h) exhibited stronger cytotoxic effect than resveratrol (IC(50) = 100 microM at 24 h) by 15-fold on human leukemic U937 cells by XTT assay. Apoptotic bodies were observed in U937 cells treated with 6 microM of heyneanol A by TUNEL assay. Heyneanol A effectively increased the portion of sub-G(1) DNA content in a time- and concentration-dependent manner by flow cytometric analysis. Heyneanol A also induced cytochrome c release from mitochondria into the cytosol and subsequent caspase activation involving caspase 9 and 3 to cleave PARP. However, it did not affect the expressions of Bax and Bcl-2 by western blotting. It was confirmed that the activation of caspase 8, 9 and 3 and the cleavage of PARP by heyneanol A were completely blocked by adding Z-VAD-FMK, a caspase inhibitor. These findings suggest that heyneanol A has anti-tumor activity, which may be mediated by apoptosis caused by cytochrome c release and caspase activation in human leukemic U937 cells.     

Use of flow cytometry for enumeration of Mycoplasma mycoides subsp. mycoides large-colony type in broth medium

Aims:  The potential of using flow cytometry (FC) in combination with a fluorescent dye (SYBR green-I) for rapidly estimating Mycoplasma mycoides subSPS. mycoides large-colony type (MmmLC) in broth culture was examined.
Methods and Results:  The FC analysis was performed by staining the MmmLC cells with a fluorescent dye, SYBR green-I (SYBR), and the results were compared with plate count method (colony forming units, – CFUs). There was a good correlation (linear regression, r ² = 0·93) between mycoplasma counts determined by FC (cells ml⁻¹) and by traditional plate count method (CFU ml⁻¹). The lowest bacterial concentration detected by FC and traditional plate count was of the order of 10⁴ cells ml⁻¹ and 10³ CFU ml⁻¹, respectively. FC method allowed results in 20–30 min, whereas at least 24 h were necessary to obtain results with the traditional plate count method (CFU).
Conclusion:  Growth rates of MmmLC in broth medium determined by FC were highly reproducible and correlated well with mycoplasma counts assessed by the plate count method.
Significance and Impact of the Study:  These findings suggest that FC could be a good alternative to replace other time-consuming techniques that are currently used to enumerate mycoplasma in broth medium, such as plate count method (CFU).     

Robust experimental design for optimizing the microbial inhibitor test for penicillin detection in milk

Aims:  To use experimental design techniques and a multiple logistic regression model to optimize a microbiological inhibition test with dichotomous response for the detection of Penicillin G in milk.
Methods and Results:  A 2³ × 2² robust experimental design with two replications was used. The effects of three control factors (V: culture medium volume, S: spore concentration of Geobacillus stearothermophilus , I: indicator concentration), two noise factors (Dt: diffusion time, Ip: incubation period) and their interactions were studied. The V, S, Dt, Ip factors and V × S, V × Ip, S × Ip interactions showed significant effects.
Conclusions:  The use of 100  μ l culture medium volume, 2 × 10⁵ spores ml⁻¹, 60 min diffusion time and 3 h incubation period is recommended. In these elaboration conditions, the penicillin detection limit was of 3·9  μ g l⁻¹, similar to the maximum residue limit (MRL). Of the two noise factors studied, the incubation period can be controlled by means of the culture medium volume and spore concentration.
Significance and Impact of the Study:  We were able to optimize bioassays of dichotomous response using an experimental design and logistic regression model for the detection of residues at the level of MRL, aiding in the avoidance of health problems in the consumer.     

Symbiotic effectiveness, rate of respiration and glutamine synthetase activity of sodium azide-resistant strains of Rhizobium leguminosarum biovar trifolii

Nitrogen fixing efficiency of sodium azide-resistant strains of Rhizobium leguminosarum bv. trifolii was studied in symbiosis with berseem clover plants in chillum jars. Rate of respiration and glutamine synthetase activity were tested in cultured cells and nodules, respectively. It was observed that shoot dry weight and percentage shoot nitrogen were maximum in plants inoculated with strains resistant to 15 μg ml⁻¹ sodium azide. Rate of respiration in cultured cells was lowest in strains resistant to 15 μg ml⁻¹ sodium azide and highest in strains resistant to 5 μg ml⁻¹ sodium azide. A negative correlation was observed between rate of respiration (in cultured cells) and shoot dry weight of host plants. Glutamine synthetase activity was maximum in nodule extracts of host plants inoculated with strains resistant to 5 and 10 μg ml⁻¹ sodium azide, whereas it was minimum for strains resistant to 15 μg ml⁻¹ sodium azide. Hence, resistance to low doses (15 μg ml⁻¹) of sodium azide, together with lower respiratory and glutamine synthetase activities, could be used as a potential method for isolating the symbiotically effective strains of Rh. leguminosarum bv. trifolii.