Modelling the inhibitory effect of copper sulfate on the growth of Penicillium expansum and Botrytis cinerea.

AIMS: This study aimed to investigate the effect of copper sulfate (from 0 to 8 mmol kg(-1)) on radial growth rate and lag time of two moulds responsible for vine grapes spoilage: Penicillium expansum strain 25·03 and Botrytis cinerea, strains BC1 and BC2. METHODS AND RESULTS: A new model was developed to describe tailing and shoulders in the inhibition curves. Because of tailing, the minimum inhibitory concentration (MIC), was not defined as the concentration at which no growth was observed, but as the concentration at which the lag time was infinite. The concentrations at which µ = µ(opt)/2, (Cu50), were in the range of 2·2-2·6 mmol kg(-1). Radial growth rate of P. expansum and the reciprocal of the lag time were linearly correlated (r = 0·84). In contrast, in the range 0-4 mmol kg(-1), an inhibition of growth of B. cinerea was observed whereas germination remained unaffected (i.e. the lag time was constant). In the range 4-8 mmol kg(-1) , the radial growth rate of B. cinerea was almost constant (c. 1 mm day(-1)), but germination was inhibited (i.e. the lag time was increased). CONCLUSIONS: The MIC values were 4·7 mmol kg(-1) for P. expansum, 8·2 and 7·3 mmol kg(-1) for B. cinerea strain BC1 and BC2, respectively, demonstrating that some isolates of these moulds are resistant to copper. SIGNIFICANCE AND IMPACT OF THE STUDY: Copper concentrations at 4 mmol kg(-1) would be sufficient to control the development of these isolates, but the toxicity of copper should be extended to other isolates and evaluated in vineyards.

Use of the Weibull model to describe inactivation of dry-harvested conidia of different Penicillium species by ethanol vapours.

AIMS: This study aimed at modelling the effect of ethanol vapours, in the range 0.7-7.5 kPa, on the inactivation of dry-harvested conidia of Penicillium chrysogenum, Penicillium digitatum and Penicillium italicum. METHODS AND RESULTS: Survival curves were modelled by a Weibull model: log (N/N(0)) = -1/2.303 (t/alpha)(beta). The shape parameter beta was different from one in all cases, indicating that the classical first-order kinetics approach is the exception rather than the rule. Survival curves exhibited upward concavity (beta < 1) with the notable exception of P. chrysogenum at ethanol vapour pressures 0.7 and 1.5 kPa. The scale parameter alpha (h) varied greatly depending on the ethanol vapour pressure and on the species. CONCLUSIONS: For safety reasons, it is recommended not to exceed an ethanol vapour pressure of 3.3 kPa. At 2.8 kPa, more than 4 log(10) reductions in viable conidia were achieved for all the species after 24-h exposure. SIGNIFICANCE AND IMPACT OF THE STUDY: Ethanol has GRAS status in the USA and represents an interesting alternative to fungicides. The effectiveness of ethanol vapours to inactivate dry-harvested conidia of some Penicillium was demonstrated in this study.

Influence of inoculum preparation on the growth of Penicillium chrysogenum.

AIMS: The influence of the spore preparation on subsequent fungal growth of Penicillium chrysogenum was assessed. METHODS AND RESULTS: The influence of four factors [the nature of the diluting solution (physiological water and physiological water added with Tween-80), the age of the sporulating culture (4, 8 and 12 days), the strain (737, 738 and 740) and the inoculum size (102, 103, 104 and 105 spores ml(-1)] on two responses (i.e. the radial growth rate, mu, and the lag time, lambda) was studied using an experimental screening methodology. CONCLUSIONS: The main conclusion was the strong effect of the inoculum size on lambda. In contrast, the diluting solution had no effect on both the experimental responses. In order to obtain the highest growth rates, it is recommended to use 4-day-old sporulating cultures with an inoculum size of 102 spores ml(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: There is a need for standardizing spore preparation in predictive mycology. The screening methodology is a powerful tool to determine the influence of qualitative and quantitative factors on various biological responses and can be applied widely in microbiology.

Comparison of volatile compound production in fruit body and in mycelium of Pleurotus ostreatus identified by submerged and solid-state cultures.

Comparative analyses of the production of volatile compounds by Pleurotus ostreatus JMO.95 fruit body and its corresponding mycelium grown in liquid, on agar surface, and on solid support cultures were carried out by dynamic headspace concentration using gas chromatography/mass spectrometry and gas chromatography sniffing. The aroma produced by fruit body was owing essentially to the presence of octan-3-one and, to a lesser extent, to octan-3-ol. Other compounds, such as oct-1-en-3-ol, oct-1-en, 2-methylbutanol, and alpha-pinene were also present in low concentrations. Comparison of aromatic spectra of the fruit body with that of mycelia obtained under different culture conditions indicated that the main aromatic compounds present in the P. ostreatus fruit body and mycelium were produced in the same proportions on agar surface and on solid support culture, but not under submerged conditions.

Relationship between spore germination kinetics and lag time during growth of Mucor racemosus.

AIMS: Spore germination requires microscopic observation whereas fungal growth results in a macroscopic examination. This paper aims at establishing a relationship between the percentage of germinated spores and parameters easily available from visible development. METHODS AND RESULTS: About 225 spores of Mucor racemosus were inoculated on PDA medium and incubated at 15 degrees and 25 degrees C. Germination kinetics were modelled by a logistic function. Fungal development provided two parameters, a growth rate, micro, and a lag period, lambda, defined as the slope of the straight line of the graph radius (mm) vs time (h) and the intercept of this line with the X-axis, respectively. CONCLUSIONS: It was found that the lag period coincided with the completion of the germination process, although the number of spores inoculated should be controlled carefully. SIGNIFICANCE AND IMPACT OF THE STUDY: Providing that this result can be generalized, this procedure would constitute a significant breakthrough for predicting food spoilage by moulds.

Comparison of the effects of temperature and water activity on growth rate of food spoilage moulds.

The influence of temperature (T) and water activity (aw) on the growth rate (mu) of seven moulds (Alternaria alternata, Aspergillus flavus, Cladosporium cladosporioides, Mucor racemosus, Penicillium chrysogenum, Rhizopus oryzae and Trichoderma harzianum) was assessed in suboptimal conditions. Firstly, the dependence of fungal growth on temperature, at aw 0.99, was modelled through an approach described previously for bacteria. A dimensionless growth rate variable: mu(dimalpha)=mu/mu (optalpha) depended on the following normalised temperature: T(dim)=(T-T(min))/(T(opt)- T(min)) according to a power function: mu(dimalpha)=[T(dim)]alpha, where alpha was an exponent to be estimated. Secondly, the same approach was used to describe the influence of aw on fungal growth, at the respective optimum temperatures for each mould. Similarly, mu(dimbeta)=mu/mu(optbeta) depended on the following normalised water activity: a(wdim)=(aw-a(wmin))/(a(wopt)-a(wmin)) according to a power function: mu(dimbeta)=[a(wdim)](beta). Results show: (i) for each mould, the alpha-value is significantly less than the beta-value, confirming that water activity has a greater influence than temperature on fungal development; (ii) the alpha-values and the beta-values depend on the mould; (iii) the alpha-value is less than 1 for the mesophilic mould A. flavus, whereas the other moulds are characterised by higher alpha-values ranging from 1.10 to 1.54; (iv) the mesophilic A. flavus exhibits a low beta-value, 1.50, compared to the hydrophilic T. harzianum, beta=2.44, while beta-values are within the range (1.71-2.37) for the other moulds.

Application of Doehlert design to determine the combined effects of temperature, water activity and pH on conidial germination of Penicillium chrysogenum.

AIMS: The influence of temperature, water activity and pH on the time necessary for germination of 90% of Penicillium chrysogenum conidia inoculated (T90) was determined. METHODS AND RESULTS: A new experimental device was developed for easy monitoring of the germination process. Experiments were carried out according to a Doehlert matrix at 11-31 degrees C, 0.86-0.98 water activity (a(w)) and pH 3.5-6.5. In these conditions, a second order polynomial relationship between T90 and the environmental factors was established for the different humectants used throughout this study (e.g. glycerol and sorbitol) with regression coefficients close to 0.97. CONCLUSIONS: For both humectants, the major effect of temperature and water activity on T90 was highlighted, whereas the effect of pH on T90 in these experimental conditions was not significant. The combined effect of temperature and water activity on T90 was also demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: Both the experimental set-up and the Doehlert matrix were well suited to determine the influence of environmental factors on mould germination.

A temperature-type model for describing the relationship between fungal growth and water activity.

Growth of Penicillium chrysogenum, Aspergillus flavus, Cladosporium cladosporioides and Alternaria alternata at their respective optimum temperatures was studied in Potato Dextrose Agar (PDA) medium at different water activities (a(w)) adjusted with glycerol. The growth rate (mu) was expressed as the increase in colony radius per unit of time. This paper extends the model that showed the relationship between temperature and bacterial growth rate developed by Rosso et al. [J. Theor. Biol. 162 (1993) 447] to describe the influence of a(w) on fungal development. An excellent correlation between the experimental data and the model predictions was obtained, the regression coefficients (r2) were greater than 0.990, with the exception of that for A. flavus (r2 = 0.982). In addition, the use of such a model allows predictions of the cardinal water activities: a(wmin), a(wopt) and a(wmax). The estimation of the minimum water activity (a(wmin)) was in accordance with data literature for all the moulds considered here, but seemed to be slightly underestimated for P. chrysogenum and A. flavus when compared to our experimental values. The estimations of the optimal water activity (a(wopt)) and the optimal growth rate (muopt) were in excellent agreement to the experimental results for the four moulds. Through this example, it is suggested that the same approach for modelling can be used for various microorganisms (e.g. bacteria and moulds), and different environmental parameters (e.g. temperature and water activity).

Prediction of conidial germination of Penicillium chrysogenum as influenced by temperature, water activity and pH.

AIMS: Conidial germination of Penicillium chrysogenum was carried out under operating conditions compatible with a pastries manufacturing process. METHODS AND RESULTS: A range, limited by two experimental values, was defined for each environmental factor tested: temperature (15 or 25 degrees C), water activity (0.75 or 0.85) and pH (3.5 or 5.5). A closed device was made, which maintained an equilibrium between water activity of the culture medium and atmospheric relative humidity during 25 days, to follow spore germination. The combined effects of temperature, water activity and pH on spore germination were studied by applying factorial design methodology. CONCLUSIONS: Higher rates of spore germination were associated with a high level of water activity. The incubation temperature also had a positive effect. A significant positive interaction between water activity and temperature was observed. Under these specific experimental conditions, pH did not have a significant effect on conidial germination. SIGNIFICANCE AND IMPACT OF THE STUDY: A model describing the behaviour of fungal conidia is proposed.

Gradient plate method to induce Streptococcus pyogenes resistance.

In recent years, increasing numbers of Streptococcus pyogenes (GAS) strains displaying resistance to macrolides have been reported in Finland, Japan, Asia and Spain. Antibiotic use has been shown to be a risk factor for infection with and carriage of drug-resistant streptococci. The aim of this study was to compare in-vitro development of resistance of streptococci to beta-lactams (penicillin, amoxycillin, cefotiam and cefuroxime) and erythromycin by serial passages in subinhibitory concentrations of antibiotics (subMICs) by gradient plate method. Three clinical strains of GAS were tested. Two were susceptible to erythromycin (MIC = 0.015 mg/L and 0.013 mg/L) and one resistant. Serial passages were performed daily by gradient plate method until a four-fold increase of the MIC was achieved. GAS variants obtained after serial passages in beta-lactams had MICs increased at least four-fold. They remained susceptible to these antibiotics. With erythromycin, final MICs reached intermediate and resistant level. Results obtained in this study with erythromycin are in good correlation with clinical studies showing that prior exposure to macrolides may help to facilitate the emergence of drug-resistant strains of streptococci.

In-vitro development of resistance to beta-lactam antibiotics in Streptococcus pneumoniae.

Selection of resistant mutants was induced in broth by exposing pneumococci to serial sub-inhibitory concentrations of various beta-lactam antibiotics. Aminopenicillins selected for resistance to themselves and to cephalosporins although cephalosporins tended to select for resistance to their own class, with the exception of cefixime which seems to select cross-resistant organisms.

In vitro development of resistance of Streptococcus pneumoniae to beta-lactam antibiotics.

In recent years, increasing numbers of Streptococcus pneumoniae strains displaying relative resistance to penicillin have been reported. Epidemiological studies have shown a correlation between aminopenicillin administration and resistance. We investigated the development of resistance in six strains (four sensitive and two intermediate-resistant to penicillin) by serial daily passages in subinhibitory concentrations of amoxicillin (AMX), amoxicillin + clavulanic acid (AMC), imipenem (IMP), cefixime (CFM), cefatrizine (CTZ), cefadroxil (CDX), and cefuroxime (CXM). MICs were determined by the macrodilution method in brain-heart broth for each daily passage. The number of daily passages needed to increase the MIC by a factor of 8 was achieved with AMX, AMC, and CFM for most of the strains after a mean of 24, 20, and 11 passages, respectively, and for one-third of the strains, with CDX, IMP, and CTZ after 11, 11, and 21 passages, respectively. Decreased susceptibility to breakpoints for intermediate-resistant S. pneumoniae populations was noted for all strains with CFM, AMX, and AMC after a mean of 10, 18, and 21 serial passages, respectively, and for four of five strains with IMP and CTZ after 12 and 13 passages. CTZ-, CDX-, and CXM-passaged variants had increased MIC values only for cephalosporins, while AMX-, AMC-, IMP-, and CFM-passaged variants exhibited increased MICs to all antibiotics tested. These in vitro data appear to be in agreement with epidemiological studies and warrant further exploration with respect to possible clinical implications.

Possible prevention of in vitro selection of resistant Streptococcus pneumoniae by beta-lactamase inhibitors.

The development of resistance in vitro in five strains of Streptococcus pneumoniae (3 with full susceptibility and 2 with intermediate susceptibility to penicillin) was investigated by serial passages in the presence of subinhibitory concentrations of amoxicillin and ampicillin. At the end of passaging, MICs of antibiotics for all the strains increased by a factor of four or more, reaching at least intermediate levels. MICs of cephalosporins, ampicillin and amoxicillin increased for almost all variants obtained. Similar results were obtained with amoxicillin plus clavulanic acid at a ratio of 2:1 and at a constant concentration of 2 micrograms/ml, and with ampicillin plus sulbactam at a ratio 2:1. In contrast, no significant modification of MIC was seen with ampicillin plus sulbactam at a constant concentration of 4 micrograms/ml sulbactam. These results suggest interaction of sulbactam with penicillin binding proteins as described previously for other bacterial species, and merit further investigation.

Pharmacokinetics and suction blister fluid penetration of a semisynthetic injectable streptogramin RP 59500 (RP 57669/ RP 54476).

RP 59500 is a new semisynthetic injectable streptogramin with excellent activity against most gram-positive bacteria. In order to assess its potential for the treatment of tissue infections, the pharmacokinetics and penetration into suction blister fluid were studied in a pilot phase I study in six male volunteers following a single infusion of 12 mg/kg over 1 h. Plasma and suction blister fluid concentrations were determined by microbiological assay. The mean peak concentration in plasma was 8.65 mg/l at the end of infusion. The mean plasma elimination half-life was 1.48 h. The mean peak concentration in interstitial fluid was 2.41 mg/l and was reached after 1 h in two volunteers and after 2 h in the other four. The mean percentage penetration for the interval 0-6 h was 82.5%. RP 59500 was still detectable in interstitial fluid at 6 h at a mean concentration of 0.92 +/- 0.25 mg/l. The data of this pilot study demonstrate good penetration of RP 59500 into non-inflammatory interstitial fluid.

Immunological reactivity of angiotensin II receptor antagonists: possible implications for receptor binding sites.

In the present study, we assessed the reactivity with seven anti-angiotensin II monoclonal antibodies of three nonpeptide and one peptide compounds described as selective antagonists of angiotensin II for AT1 (DuP 753, 2-n-butyl-4-chloro-5-(hydroxymethyl)-1-[[2'-(1H-tetrazol-5-yl) biphenyl-4-yl] methyl] imidazole; EXP 3174, 2-n-butyl-4-chloro-5-(carboxylic acid)-1-[[2'-(1H-tetrazol-5-yl) biphenyl-4-yl] methyl] imidazole) and AT2 receptor sites (CGP42112A, nicotinyl-Tyr-(N alpha-benzyloxycarbonyl-Arg)Lys-His-Pro-Ile-OH; PD123177, 1-[(4-amino-3-methylphenyl) methyl]-5-(diphenyl-acetyl)-4,5,6,7-tetrahydro-1H-imidazol[4,5-c] pyridine 6-carboxylic acid), respectively. These studies were undertaken because the reactivity of the monoclonal antibodies with peptide analogs of angiotensin II and the three-dimensional structure of an angiotensin II-immunoglobulin Fab fragment complex strongly suggested that the conformations identified by the monoclonal antibodies were relevant to those involved in receptor binding as defined by biophysical models supported by structure activity studies. Surprisingly although three of the compounds were described as competitive inhibitors of angiotensin II, binding of the various monoclonal antibodies to either ovalbumin-coupled angiotensin II adsorbed to plastic wells or 125I-labeled angiotensin II in liquid phase was unaffected by any of the nonpeptide antagonists and CGP42112A up to 10(-4) M concentration. The antagonists also failed to bind to rabbit polyclonal anti-angiotensin II antibodies. Direct binding experiments in which solid phase-immobilized angiotensin II and DuP 753 conjugates were incubated with anti-angiotensin II or anti-DuP 753 monoclonal antibodies, did not show any cross-reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of Linoleic Acid or Mevalonate and 6-Pentyl-alpha-Pyrone Biosynthesis by Trichoderma Species.

The understanding of the biosynthetic pathway of 6-pentyl-alpha-pyrone in Trichoderma species was achieved by using labelled linoleic acid or mevalonate as a tracer. Incubation of growing cultures of Trichoderma harzianum and T. viride with [U-C]linoleic acid or [5-C]sodium mevalonate revealed that both fungal strains were able to incorporate these labelled compounds (50 and 15%, respectively). Most intracellular radioactivity was found in the neutral lipid fraction. At the initial time of incubation, the radioactivity from [C]linoleic acid was incorporated into 6-pentyl-alpha-pyrone more rapidly than that from [C]mevalonate. No radioactivity incorporation was detected in 6-pentyl-alpha-pyrone when fungal cultures were incubated with [1-C]linoleic acid. These results suggested that beta-oxidation of linoleic acid was a probable main step in the biosynthetic pathway of 6-pentyl-alpha-pyrone in Trichoderma species.