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PubMed1998

Sampling the light-organ microenvironment of Euprymna scolopes: description of a population of host cells in association with the bacterial symbiont Vibrio fischeri.

The symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri has a pronounced diel rhythm, one component of which is the venting of the contents of the light organ into the surrounding seawater each day at dawn. In this study, we explored the use of this behavior to sample the microenvironment of the light-organ crypts. Intact crypt contents, which emerge from the lateral pores of the organ as a thick paste-like exudate, were collected from anesthetized host animals that had been exposed to a light cue. Microscopy revealed that the expelled material is composed of a conspicuous population of host cells in association with the bacterial symbionts, all of which are embedded in a dense acellular matrix that strongly resembles the bacteria-based biofilms described in other systems. Assays of the viability of expelled crypt cells revealed no dead bacterial symbionts and a mixture of live and dead host cells. Analyses of the ultrastructure, biochemistry, and phagocytic activity of a subset of the host cell population suggested that some of these cells are macrophage-like molluscan hemocytes.

Animals↗

PubMed1961

Possible effects of antibiotic treatment on the senstivity and growth requirements of Neisseria gonorrhoea.

The decreased sensitivity to penicillin of Neisseria gonorrhoeae has been a cause of concern during recent years. About 25% of strains sent in for routine diagnosis to the Statens Seruminstitut in 1957 were found to require roughly 20 times as much penicillin to inhibit development completely as was required for strains isolated in 1944. The stability of this reduced sensitivity to penicillin was studied in 20 strains which were subcultured daily on medium without penicillin. A comparison was also made between the sensitivity levels to penicillin and streptomycin for 1957 and 1958. A phenomenon possibly resulting from penicillin therapy of gonorrhoea was noted by the authors in June 1957, when they recorded the appearance of atypical strains having special growth requirements, low viability and weak or absent glucose-fermentation. The incidence, serology and sensitivity to antibiotics of these atypical strains were examined; further research on their different behaviour in response to changes in medium, as compared with that of typical strains, may give rise to some interesting findings.

Anti-Bacterial Agents↗

PubMed2000

Effects of fructooligosaccharides and their monomeric components on bile salt resistance in three species of bifidobacteria.

The influence of fructooligosaccharides (FOS) and their monomeric components on bile salt resistance of Bifidobacterium breve ATCC 15700, Bif. longum ATCC 15707 and Bif. animalis ATCC 25527 was examined. The neosugars induced fructofuranosidase activities for the degradation of these saccharides. For the three strains tested the growth was identical and bile salts had the same inhibitory effect on growth whatever the carbohydrate used. The survival of Bif. breve and Bif. longum, in the presence of glycodeoxycholic acid depended, however, on carbohydrates: the toxic effects of the bile salt could be partly alleviated by the addition of a metabolizable C-source. For Bif. animalis, the presence of any carbohydrate in the incubation medium did not enhance the viability of the strain. But in the three deconjugating strains of bifidobacteria studied, the presence of neosugar during the growth led to improved resistance to the bactericidal effect of the bile salt compared with the monomeric components of these neosugars (glucose and fructose).

Animals↗

PubMed2005

Lactobacillus johnsonii La1 attenuates Helicobacter pylori-associated gastritis and reduces levels of proinflammatory chemokines in C57BL/6 mice.

In clinical settings, Lactobacillus johnsonii La1 administration has been reported to have a favorable effect on Helicobacter pylori-associated gastritis, although the mechanism remains unclear. We administered, continuously through the water supply, live La1 to H. pylori-infected C57BL/6 mice and followed colonization, the development of H. pylori-associated gastritis in the lamina propria, and the levels of proinflammatory chemokines macrophage inflammatory protein 2 (MIP-2) and keratinocyte-derived cytokine (KC) in the serum and gastric tissue over a period of 3 months. We documented a significant attenuation in both lymphocytic (P=0.038) and neutrophilic (P=0.003) inflammatory infiltration in the lamina propria as well as in the circulating levels of anti-H. pylori immunoglobulin G antibodies (P=0.003), although we did not observe a suppressive effect of La1 on H. pylori colonizing numbers. Other lactobacilli, such as L. amylovorus DCE 471 and L. acidophilus IBB 801, did not attenuate H. pylori-associated gastritis to the same extent. MIP-2 serum levels were distinctly reduced during the early stages of H. pylori infection in the La1-treated animals, as were gastric mucosal levels of MIP-2 and KC. Finally, we also observed a significant reduction (P=0.046) in H. pylori-induced interleukin-8 secretion by human adenocarcinoma AGS cells in vitro in the presence of neutralized (pH 6.8) La1 spent culture supernatants, without concomitant loss of H. pylori viability. These observations suggest that during the early infection stages, administration of La1 can attenuate H. pylori-induced gastritis in vivo, possibly by reducing proinflammatory chemotactic signals responsible for the recruitment of lymphocytes and neutrophils in the lamina propria.

Animals↗

PubMed2005

A review of technologies for rapid detection of bacteria in recreational waters.

Monitoring of recreational beaches for fecal indicator bacteria is currently performed using culture-based technology that can require more than a day for laboratory analysis, during which time swimmers are at risk. Here we review new methods that have the potential to reduce the measurement period to less than an hour. These methods generally involve two steps. The first is target capture, in which the microbial group of interest (or some molecular/chemical/or biochemical signature of the group) is removed, tagged or amplified to differentiate it from the remaining material in the sample. We discuss three classes of capture methods: 1) Surface and whole-cell recognition methods, including immunoassay techniques and molecule-specific probes; 2) Nucleic acid methods, including polymerase chain reaction (PCR), quantitative PCR (Q-PCR), nucleic acid sequence based amplification (NASBA) and microarrays; and 3) Enzyme/substrate methods utilizing chromogenic or fluorogenic substrates. The second step is detection, in which optical, electrochemical or piezoelectric technologies are used to quantify the captured, tagged or amplified material. The biggest technological hurdle for all of these methods is sensitivity, as EPA's recommended bathing water standard is less than one cell per ml and most detection technologies measure sample volumes less than 1 ml. This challenge is being overcome through addition of preconcentration or enrichment steps, which have the potential to boost sensitivity without the need to develop new detector technology. The second hurdle is demonstrating a relationship to health risk, since most new methods are based on measuring cell structure without assessing viability and may not relate to current water quality standards that were developed in epidemiology studies using culture-based methods. Enzyme/substrate methods may be the first rapid methods adopted because they are based on the same capture technology as currently-approved EPA methods and their relationship to health risk can be established by demonstrating equivalency to existing procedures. Demonstration of equivalency may also be possible for some surface and whole-cell recognition methods that capture bacteria in a potentially viable state. Nucleic acid technologies are the most versatile, but measure nonviable structure and will require inclusion in epidemiological studies to link their measurement with health risk.

Bacteria↗

PubMed1998

Characterization of organotin-resistant bacteria from boston harbor sediments

Organotins are widely used in agriculture and industry. They are toxic to a variety of organisms including bacteria, although little is known of their physiology and ecology. Bacteria resistant to six organotins-tributyltin (TBT), dibutyltin (DBT), monobutyltin (MBT), triphenyltin (TPT), diphenyltin (DPT), and monophenyltin (MPT)-were isolated from Boston Harbor sediments, Massachusetts, USA. Bacteria resistant to each of the organotins, except DPT, were isolated directly from estuarine sediments. Viability of the organotin-resistant bacteria on serial transfer in the laboratory ranged from 80 to 91%. Each isolate was screened for resistance to the other organotins. All of 250 isolates were resistant to at least two organotins. No DPT-resistant isolates were found on initial isolation on DPT, although there was DPT resistance among the other organotin-resistant bacteria. Eighty percent of TBT-resistant bacteria were TPT-resistant, suggesting that antifouling paints containing TPT will not be a suitable substitute for TBT in paints designed to inhibit microbial biofilms. Debutylation reduced toxicity in some cases while dephenylation did not. Thus, even though trisubstituted organotins are generally believed to be more toxic than di- or monosubstituted organotins, this may not always be the case, and more than one mechanism of resistance may be involved. All the bacteria were resistant to at least six of eight heavy metals tested, suggesting that resistance to heavy metals may be associated with resistance to organotins.

Journal Article↗

PubMed2006

Survival of Listeria monocytogenes Scott A on metal surfaces: implications for cross-contamination.

Listeria monocytogenes is an important re-emerging pathogen which is commonly found in the environment. Many outbreaks have been associated with the contamination of food produce, often linked to cross-contamination from surfaces or equipment to prepared foodstuffs. In the present study a number of copper-base metal alloys have been used to assess the survival times of L. monocytogenes on different materials, in comparison with stainless steel. High concentrations (10(7)) of bacteria were placed on metal coupons cut from each alloy. After defined incubation times, coupons were placed in tubes containing phosphate buffered saline and vortexed to remove the cells. Aliquots were then plated onto tryptone blood agar plates and the number of colony forming units counted. The high concentration of bacteria was used to represent a "worst-case" scenario. The results indicate that survival is greatly reduced on a copper-base alloy compared to stainless steel. Viable cells could be detected on stainless steel after 24 h incubation at room temperature. On copper, brass, aluminium bronze and silicon bronze, no viable bacteria could be detected after 60 min incubation, indicating a 5 log reduction (the detection limit of the procedure was 100 bacteria). No cells could be detected from copper nickel and copper nickel zinc alloys, after 90 min incubation. The viability stain, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), confirmed these results, with actively respiring bacteria being clearly labelled on stainless steel after 24 h. The results suggest that careful choice of surface material could reduce the potential risk of cross-contamination in industrial, commercial and domestic environments.

Alloys↗

PubMed1989

A preliminary assessment of the feasibility of evaluating promising antifilarials in vitro against adult Onchocerca volvulus.

The suitability of motility indices and tetrazolium-based colorimetric assays for the determination of the viability of adult Onchocerca volvulus after in vitro exposure to potential macrofilaricides has been examined. Experimentation showed that both techniques could be applied to adult O. volvulus, although the variability between individual worms necessitated the use of large experimental groups. The potential of using cut anterior tips of female O. volvulus for screening was also investigated. These were shown to give reasonably consistent motility indices, and drug effects were discernible even after 72 h in vitro culture. Application of these viability criteria to studies on the short-term in vitro survival of intact male and female O. volvulus incubated in Eagles MEM plus serum, under 5% CO2 in air, showed this medium to be suboptimal with a greater than 50% loss of worm viability within 144 h of nodulectomy. Males isolated by the collagenase technique were shown to be significantly less viable than dissected males, by both motility indices and tetrazolium reduction. The results highlight the need to use either dissected males, or in the case of females, the need to minimize exposure to collagenase solution. A possible mechanism for selecting a more uniformly viable female worm population is discussed. Examination of the in vitro effects of CGP 20376 using these viability criteria/assay systems showed some delayed suppression of worm motility, but after 120 h in vitro CGP 20376 was not macrofilaricidal against male or female O. volvulus. Male worms were also implanted subcutaneously into gerbils.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2003

Calcineurin A of Candida albicans: involvement in antifungal tolerance, cell morphogenesis and virulence.

The azole antifungal fluconazole possesses only fungistatic activity in Candida albicans and, therefore, this human pathogen is tolerant to this agent. However, tolerance to fluconazole can be inhibited when C. albicans is exposed to fluconazole combined with the immunosuppressive drug cyclosporin A, which is known to inhibit calcineurin activity in yeast. A mutant lacking both alleles of a gene encoding the calcineurin A subunit (CNA) lost viability in the presence of fluconazole, thus making calcineurin essential for fluconazole tolerance. Consistent with this observation, tolerance to fluconazole was modulated by calcium ions or by the expression of a calcineurin A derivative autoactivated by the removal of its C-terminal inhibitory domain. Interestingly, CNA was also essential for tolerance to other antifungal agents (voriconazole, itraconazole, terbinafine, amorolfine) and to several other metabolic inhibitors (caffeine, brefeldin A, mycophenolic acid, fluphenazine) or cell wall-perturbing agents (SDS, calcofluor white, Congo red), thus indicating that the calcineurin pathway plays an important role in the survival of C. albicans in the presence of external growth inhibitors. Several genes, including PMC1, a vacuolar calcium P-type ATPase, were regulated in a calcineurin- and fluconazole-dependent manner. However, PMC1 did not play a direct role in the survival of C. albicans when exposed to fluconazole. In addition to these different properties, calcineurin was found to affect colony morphology in several media known to modulate the C. albicans dimorphic switch. In particular, calcineurin was found to be essential for C. albicans viability in serum-containing media. Finally, calcineurin was found to be necessary for the virulence of C. albicans in a mice model of infection, thus making calcineurin an important element for adequate adaptation to the conditions of the host environment.

Antifungal Agents↗

PubMed2006

Differential gene expression in response to phenol and catechol reveals different metabolic activities for the degradation of aromatic compounds in Bacillus subtilis.

Aromatic organic compounds that are present in the environment can have toxic effects or provide carbon sources for bacteria. We report here the global response of Bacillus subtilis 168 to phenol and catechol using proteome and transcriptome analyses. Phenol induced the HrcA, sigmaB and CtsR heat-shock regulons as well as the Spx disulfide stress regulon. Catechol caused the activation of the HrcA and CtsR heat-shock regulons and a thiol-specific oxidative stress response involving the Spx, PerR and FurR regulons but no induction of the sigmaB regulon. The most surprising result was that several catabolite-controlled genes are derepressed by catechol, even if glucose is taken up under these conditions. This derepression of the carbon catabolite control was dependent on the glucose concentration in the medium, as glucose excess increased the derepression of the CcpA-dependent lichenin utilization licBCAH operon and the ribose metabolism rbsRKDACB operon by catechol. Growth and viability experiments with catechol as sole carbon source suggested that B. subtilis is not able to utilize catechol as a carbon-energy source. In addition, the microarray results revealed the very strong induction of the yfiDE operon by catechol of which the yfiE gene shares similarities to glyoxalases/bleomycin resistance proteins/extradiol dioxygenases. Using recombinant His6-YfiE(Bs) we demonstrate that YfiE shows catechol-2,3-dioxygenase activity in the presence of catechol as the metabolite 2-hydroxymuconic semialdehyde was measured. Furthermore, both genes of the yfiDE operon are essential for the growth and viability of B. subtilis in the presence of catechol. Thus, our studies revealed that the catechol-2,3-dioxygenase YfiE is the key enzyme of a meta cleavage pathway in B. subtilis involved in the catabolism of catechol.

Bacillus subtilis↗

PubMed2005

Divalent metal cations increase the activity of the antimicrobial Peptide kappacin.

Kappacin, nonglycosylated kappa-casein(106-169), is a novel antimicrobial peptide produced from kappa-casein found in bovine milk. There are two major genetic forms of kappacin, A and B, and using synthetic peptides corresponding to the active region, kappa-casein(138-158), of these forms, we have shown that the Asp148 to Ala148 substitution is responsible for the lesser antibacterial activity of kappa-casein-B(106-169). Kappacin was shown to have membranolytic action at concentrations above 30 microM at acidic pH when tested against artificial liposomes. There was little membranolytic activity at neutral pH, which is consistent with the lack of antibacterial activity of kappacin against Streptococcus mutans at this pH. Kappacin specifically bound two zinc or calcium ions per mol, and this binding enhanced antibacterial activity at neutral pH. Nuclear magnetic resonance analysis indicated that a kappa-casein-A(138-158) synthetic peptide undergoes a conformational change in the presence of the membrane solvent trifluoroethanol and excess divalent metal ions. This change in conformation is presumably responsible for the increase in antibacterial activity of kappacin detected in the presence of excess zinc or calcium ions at neutral pH. When tested against the oral bacterial pathogen S. mutans cultured as a biofilm in a constant-depth film fermentor, a preparation of 10 g/liter kappacin and 20 mM ZnCl2 reduced bacterial viability by 3 log10 and suppressed recovery of viability. In contrast 20 mM ZnCl2 alone reduced bacterial viability by approximately 1 log10 followed by rapid recovery. In conclusion, kappacin has a membranolytic, antibacterial effect that is enhanced by the presence of divalent cations.

Anti-Bacterial Agents↗

PubMed2004

Regulation of the intracellular free iron pool by Dpr provides oxygen tolerance to Streptococcus mutans.

Dpr is an iron-binding protein required for oxygen tolerance in Streptococcus mutans. We previously proposed that Dpr could confer oxygen tolerance to the bacterium by sequestering intracellular free iron ions that catalyze generation of highly toxic radicals (Y. Yamamoto, M. Higuchi, L. B. Poole, and Y. Kamio, J. Bacteriol. 182:3740-3747, 2000; Y. Yamamoto, L. B. Poole, R. R. Hantgan, and Y. Kamio, J. Bacteriol. 184:2931-2939, 2002). Here, we examined the intracellular free iron status of wild-type (WT) and dpr mutant strains of S. mutans, before and after exposure to air, by using electron spin resonance spectrometry. Under anaerobic conditions, free iron ion concentrations of WT and dpr strains were 225.9 +/- 2.6 and 333.0 +/- 61.3 microM, respectively. Exposure of WT cells to air for 1 h induced Dpr expression and reduced intracellular free iron ion concentrations to 22.5 +/- 5.3 microM; under these conditions, dpr mutant cells maintained intracellular iron concentration at 230.3 +/- 28.8 microM. A decrease in cell viability and genomic DNA degradation was observed in the dpr mutant exposed to air. These data indicate that regulation of the intracellular free iron pool by Dpr is required for oxygen tolerance in S. mutans.

Adaptation, Physiological↗

PubMed1976

Enzymatic dissociation and short-term culture of isolated rat anterior pituitary cells for studies on the control of hormone secretion.

A convenient procedure has been developed for preparing a suspension of isolated rat anterior pituitary cells which retains responsiveness to secretagogues. Rat anterior pituitaries were dispersed with collagenase and hyaluronidase followed by mechanical dispersion by means of a Pasteur pipette. Immediately after dispersion, the cells showed only slight responses to secretagogues, whereas after short-term culture (20-22 h) in the presence of sera, the cells recovered their ability to respond to synthetic LH-releasing hormone (LHRH) and synthetic thyrotropin-releasing hormone (TRH). During a 3-h incubation, cells prepared from pituitaries of male rats released LH and FSH, or TSH and prolactin (PRL) in amounts directly related to the dose of synthetic LHRH or TRH, respectively. The minimum effective concentrations of hypophysiotropic hormones lay between 10(-10) and 10(-9)M, although it was observed that cells originating from female rats usually gave quicker and larger responses to LHRH. No significant net increase in the total hormonal content (cells + medium) of radioimmunoassayable LH or FSH in response to LHRH, or of TSH or PRL in response to TRH, was observed during the 3-h incubation period. The cells released significant amounts of PRL, TSH, and to a lesser extent, LH, in response to 1-5 X 10-3M N6,O2'-dibutyryl cyclic AMP, accompanied by remarkable elevation in total content (cells + medium) of PRL and TSH but not of LH. The response of the cells to theophylline or high [K+] was similar to that usually observed in previous hemipituitary experiments. These results demonstrate the viability of this in vitro cell system and its suitability for further study of the regulation of the secretion of pituitary hormones.

Animals↗

PubMed1987

[Biological properties of Mycoplasma isolated from bulls' semen].

The occurrence of Mycoplasma in bulls' semen obtained from different Artificial Insemination Centers in Poland was studied. Out of 380 semen samples 234 mycoplasma strains were isolated (61%). Nutritional requirements and biochemical properties of the isolated strains were studied. On the basis of the results of catabolism of glucose and arginine three different groups of isolates were found. The final identification was determined by growth inhibition test and indirect antibody fluorescent technique. 98% of the isolated strains were classified. In Acholeplasma genus two species were found: A. laidlawii and A. modicum. In the genus of Mycoplasma the majority of isolates were identified as M. bovigenitalium (76%). The remaining strains belonged to three other species: M. bovis, M. bovirhinis, M. arginini and to serological group number 7. The sensitivity of isolates to streptomycin and their viability at different temperature were examined. The occurrence of the pathogenic Mycoplasma strains in the examined bulls' semen, their long survival time at -196 degrees C (24 months) and the resistance of some strains to streptomycin indicate the necessity to examine the semen before it is used in artificial insemination.

Animals↗

PubMed2003

Susceptibility of oral bacteria to an antimicrobial decapeptide.

Naturally occurring antimicrobial peptides have emerged as alternative classes of antimicrobials. In general, these antimicrobial peptides exhibit selectivity for prokaryotes and minimize the problems of engendering microbial resistance. As an alternative method to search for more effective broad-spectrum peptide antimicrobials, investigators have developed peptide libraries by using synthetic combinatorial technology. A novel decapeptide, KKVVFKVKFK (KSL), has been identified that shows a broad range of antibacterial activity. The purpose of this study was to test the efficacy of this antimicrobial peptide in killing selected strains of oral pathogens and resident saliva bacteria collected from human subjects. Cytotoxic activity of KSL against mammalian cells and the structural features of this decapeptide were also investigated, the latter by using two-dimensional NMR in aqueous and DMSO solutions. MICs of KSL for the majority of oral bacteria tested in vitro ranged from 3 to 100 microg ml(-1). Minimal bactericidal concentrations of KSL were, in general, within one to two dilutions of the MICs. KSL exhibited an ED(99) (the dose at which 99 % killing was observed after 15 min at 37 degrees C) of 6.25 microg ml(-1) against selected strains of Lactobacillus salivarius, Streptococcus mutans, Streptococcus gordonii and Actinobacillus actinomycetemcomitans. In addition, KSL damaged bacterial cell membranes and caused 1.05 log units reduction of viability counts of saliva bacteria. In vitro toxicity studies showed that KSL, at concentrations up to 1 mg ml(-1), did not induce cell death or compromise the membrane integrity of human gingival fibroblasts. NMR studies suggest that KSL adopts an alpha-helical structure in DMSO solution, which mimics the polar aprotic membrane environment, whereas it remains unstructured in aqueous medium. This study shows that KSL may be a useful antimicrobial agent for inhibiting the growth of oral bacteria that are associated with caries development and early plaque formation.

Aggregatibacter actinomycetemcomitans↗

PubMed2002

Microbial survival of space vacuum and extreme ultraviolet irradiation: strain isolation and analysis during a rocket flight.

We have recovered new isolates from hot springs, in Yellowstone National Park and the Kamchatka Peninsula, after gamma-irradiation and exposure to high vacuum (10(-6) Pa) of the water and sediment samples. The resistance to desiccation and ionizing radiation of one of the isolates, Bacillus sp. strain PS3D, was compared to that of the mesophilic bacterium, Deinococcus radiodurans, a species well known for its extraordinary resistance to desiccation and high doses of ionizing radiation. Survival of these two microorganisms was determined in real and simulated space conditions, including exposure to extreme UV radiation (10-100 nm) during a rocket flight. We found that up to 15 days of desiccation alone had little effect on the viability of either bacterium. In contrast, exposure to space vacuum ( approximately 10(-6) Pa) decreased cell survival by two and four orders of magnitude for Bacillus sp. strain PS3D and D. radiodurans, respectively. Simultaneous exposure to space vacuum and extreme UV radiation further decreased the survival of both organisms, compared to unirradiated controls. This is the first report on the isolated effect of extreme UV at 30 nm on cell survival. Extreme UV can only be transmitted through high vacuum, therefore its penetration into the cells may only be superficial, suggesting that in contrast to near UV, membrane proteins rather than DNA were damaged by the radiation.

Bacillus↗

PubMed2006

Antimicrobial activity of potato aspartic proteases (StAPs) involves membrane permeabilization.

Solanum tuberosum aspartic proteases (StAPs) with antimicrobial activity are induced after abiotic and biotic stress. In this study the ability of StAPs to produce a direct antimicrobial effect was investigated. Viability assays demonstrated that StAPs are able to kill spores of Fusarium solani and Phytophthora infestans in a dose-dependent manner. Localization experiments with FITC-labelled StAPs proved that the proteins interact directly with the surface of spores and hyphae of F. solani and P. infestans. Moreover, incubation of spores and hyphae with StAPs resulted in membrane permeabilization, as shown by the uptake of the fluorescent dye SYTOX Green. It is concluded that the antimicrobial effect of StAPs against F. solani and P. infestans is caused by a direct interaction with the microbial surfaces followed by membrane permeabilization.

Antifungal Agents↗

PubMed1992

Evidence of reduced poly-B-hydroxybutyrate biosynthesis in free-living nitrogen-fixing bacteria, Azotobacter chroococcum, following acquired resistance to the fungicide captan.

Some biological activities of Azotobacter chroococcum, strain Azcap 1, (spontaneous mutant, captan resistant up to 300 micrograms/ml) were assayed on RM medium with and without the presence of the fungicide. Comparisons were also carried out with Az. chroococcum sensitive strains Azwt, Azcan 10 and 14. The hydrolysis of captan, incorporated in agar plates of RM at 100 micrograms/ml, was rapid, since on 4-day plates, no effect was found on the strain Azwt, while on freshly prepared ones its growth was completely blocked. As for Azcap 1, grown on RM only, the behaviour was similar to that of sensitive strains, whereas when grown on captan the results of experiments showed: (i) a lag of approximately 12 h to reach the maximum nitrogen-fixing activity; (ii) delay of 12-24 h in the full consumption of glucose present in the medium, although the invertase activity did not present differences; (iii) high ATP culture content during the 50 h of the experiment; (iv) approximately 6-10-fold lower production of PHB (poly-B-hydroxybutyrate); (v) lack of typical encystment phase, for the tested 96 h and reduced viability in developing colonies on agar RM medium. In contrast, when captan was added to cultural medium at sublethal concentration, 50 micrograms/ml for sensitive strain Azwt and 200 micrograms/ml for Azcap 1, the amount of glutathione produced (to remove the fungicide toxicity) was several times higher for the former.

Azotobacter↗