Survival of Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria in soil

The survival of mesophilic Aeromonas spp. in soil in the presence or absence of indigenous microflora was evaluated in a laboratory study. Two cytotoxic ( Aer. hydrophila and Aer. caviae ) and one invasive ( Aer. sobria ) clinical isolate strains were selected for this study. After contamination of sterile or unsterilized soil with the three strains of Aeromonas , the number of living cells was determined over at least 5 months. For all strains the survival curves were characterized by an initial re-growth followed by a slow inactivation of bacteria, with significant differences due to the presence of indigenous microflora. The times necessary to achieve a 95% reduction of the initial population were > 140, 113 and 62 d in sterilized soil respectively for Aer. caviae, Aer. hydrophila and Aer. sobria , while the corresponding times in unsterilized soil were 42, 38 and 11 d. All strains preserved the virulence factors for the entire period of the study. These results suggest that the soil may be an important reservoir for Aeromonas spp. and, thus, may play an important role in the epidemiology of Aeromonas -associated human infections.     

Towards MRI contrast agents of improved efficacy. NMR relaxometric investigations of the binding interaction to HSA of a novel heptadentate macrocyclic triphosphonate Gd(III)-complex

 A novel heptacoordinating ligand consisting of a thirteen-membered tetraazamacrocycle containing the pyridine ring and bearing three methylenephosphonate groups (PCTP-[13]) has been synthesized. Its Gd(III) complex displays a remarkably high longitudinal water proton relaxivity (7.7 mM^–1 s^–1 at 25  °C, 20 MHz and pH 7.5) which has been accounted for in terms of contributions arising from (1) one water molecule bound to the metal ion, (2) hydrogen-bonded water molecules in the second coordination sphere, or (3) water molecules diffusing near the paramagnetic chelate. Variable-temperature ¹⁷O-NMR transverse relaxation data indicate that the residence lifetime of the metal-bound water molecule is very short (8.0 ns at 25  °C) with respect to the Gd(III) complexes currently considered as contrast agents for magnetic resonance imaging. Furthermore, GdPCTP-[13] interacts with human serum albumin (HSA), likely through electrostatic forces. By comparing water proton relaxivity data for the GdPCTP-[13]-HSA adduct, measured as a function of temperature and magnetic field strength, with those for the analogous adduct with GdDOTP (a twelve-membered tetraaza macrocyclic tetramethylenephosphonate complex lacking a metal-bound water molecule), it has been possible to propose a general picture accounting for the main determinants of the relaxation enhancement observed when a paramagnetic Gd(III) complex is bound to HSA. Basically, the relaxation enhancement in these systems arises from (1) water molecules in the hydration shell of the macromolecule and protein exchangeable protons which lie close to the interaction site of the paramagnetic complex and (2) the metal bound water molecule(s). As far as the latter contribution is concerned, the interaction with the protein causes an elongation of the residence lifetime of the metal-bound water molecule, which limits, to some extent, the potential relaxivity enhancement expected upon the binding of the paramagnetic complex to HSA. Received: 27 January 1997 / Accepted: 12 May 1997     

A rapid mini-prep method for isolation of ectomycorrhizal DNA from Tuber species

A rapid procedure has been developed to isolate DNA from the ectomycorrhizae of Tuber spp. for use in PCR experiments. The method described is fast and sensitive and can overcome the amplification problems that can arise in the presence of inhibitors. For this reason it can be used to type ectomycorrhizae even starting from a single root tip and make mycorrhizae identification much more rapid.     

Survival ability of cytotoxic strains of motile Aeromonas spp. in different types of water

The ability of motile Aeromonas spp. to survive in drinking water (mineral and tap water) and in sea water was experimentally tested. Clinically isolated cytotoxic strains of A. hydrophila, A. caviae and A. sobria were selected for this study. After contamination of water samples, the survival of Aeromonas strains was studied for at least three months using viable counts. The results obtained show that the survival of the Aeromonas spp. varies considerably depending on species and water type. For all three species, the survival time was longest in mineral water, where viable bacteria of each strain were still detected after 100 d. Moreover, A hydrophila and A. caviae also re-grew on the first day. In tap water all strains showed marked survival, although to a lesser extent than in mineral water. Aeromonas cells showed a rapid decline in sea water (90% reduction in viable cells after about two d) and thus seem to be more sensitive to saline/marine stress than chlorination.     

Antifungal properties of the human Metschnikowia strain IHEM 25107

Metschnikowiaceae may show natural, strain-dependent antifungal properties, so they are employed in agriculture as natural, safe alternatives to pesticides. With this paper, we documented the ability of the recently described Metschnikowia IHEM 25107 to inhibit growth of several molds, including diverse Aspergillus species and dermatophytes. This biocontrol activity enables pulcherrimin-producing strains to naturally antagonize competing microorganisms.     

Molecular characterisation of the small GTPase CDC42 in the ectomycorrhizal fungus Tuber borchii Vittad.

Summary. The small GTPase CDC42 is ubiquitously expressed in eukaryotes, where it participates in the regulation of the cytoskeleton and a wide range of cellular processes, including cytokinesis, gene expression, cell cycle progression, apoptosis, and tumorigenesis. As very little is known on the molecular level about mycorrhizal morphogenesis and development and these events depend on a tightly regulated reorganisation of the cytoskeleton network in filamentous fungi, we focused on the molecular characterisation of the cdc42 gene in Tuber borchii Vittad., an ascomycetous hypogeous fungus forming ectomycorrhizae. The entire gene was isolated from a T. borchii cDNA library and Southern blot analyses showed that only one copy of cdc42 is present in the T. borchii genome. The predicted amino acid sequence is very similar to those of other known small GTPases and the similar domain structures suggest a similar function. Real-time PCR analyses revealed an increased expression of Tbcdc42 during the phase preparative to the instauration of symbiosis, in particular after stimulation with root exudate extracts. Immunolocalisation experiments revealed an accumulation of CDC42 in the apical tips of the growing hyphae. When a constitutively active Tbcdc42 mutant was expressed in Saccharomyces cerevisiae, morphological changes typical of pseudohyphal growth were observed. Our results suggest a fundamental role of CDC42 in cell polarity development in T. borchii. Correspondence and reprints: Istituto di Chimica Biologica “G. Fornaini”, Università degli Studi di Urbino, Via Saffi 2, 61029 Urbino, Italy.     

Testing multiple coordination constraints with a novel bimanual visuomotor task

The acquisition of a new bimanual skill depends on several motor coordination constraints. To date, coordination constraints have often been tested relatively independently of one another, particularly with respect to isofrequency and multifrequency rhythms. Here, we used a new paradigm to test the interaction of multiple coordination constraints. Coordination constraints that were tested included temporal complexity, directionality, muscle grouping, and hand dominance. Twenty-two healthy young adults performed a bimanual dial rotation task that required left and right hand coordination to track a moving target on a computer monitor. Two groups were compared, either with or without four days of practice with augmented visual feedback. Four directional patterns were tested such that both hands moved either rightward (clockwise), leftward (counterclockwise), inward or outward relative to each other. Seven frequency ratios (3∶1, 2∶1, 3∶2, 1∶1, 2∶3. 1∶2, 1∶3) between the left and right hand were introduced. As expected, isofrequency patterns (1∶1) were performed more successfully than multifrequency patterns (non 1∶1). In addition, performance was more accurate when participants were required to move faster with the dominant right hand (1∶3, 1∶2 and 2∶3) than with the non-dominant left hand (3∶1, 2∶1, 3∶2). Interestingly, performance deteriorated as the relative angular velocity between the two hands increased, regardless of whether the required frequency ratio was an integer or non-integer. This contrasted with previous finger tapping research where the integer ratios generally led to less error than the non-integer ratios. We suggest that this is due to the different movement topologies that are required of each paradigm. Overall, we found that this visuomotor task was useful for testing the interaction of multiple coordination constraints as well as the release from these constraints with practice in the presence of augmented visual feedback.     

Determination of the Viability of Aeromonas hydrophila in Different Types of Water by Flow Cytometry, and Comparison with Classical Methods

The presence of Aeromonas spp. in water can represent a risk for human health. Therefore, it is important to know the physiological status of these bacteria and their survival in the environment. We studied the behavior of a strain of Aeromonas hydrophila in river water, spring water, brackish water, mineral water, and chlorinated drinking water, which had different physical and chemical characteristics. The bacterial content was evaluated by spectrophotometric and plate count techniques. Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria. The traditional methods showed that the bacterial content was variable and dependent on the type of water. The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. According to our results, flow cytometry is a suitable method for assessing the viability of bacteria in water samples. Furthermore, it permits fast detection of bacteria that are in a viable but nonculturable state, which are not detectable by conventional methods.     

Modifications of Bordetella bronchiseptica core lipopolysaccharide influence immune response without affecting protective activity

Bordetella bronchiseptica produces respiratory disease primarily in mammals including humans. Although a considerably amount of research has been generated regarding lipopolysaccharide (LPS) role during infection and stimulating innate and adaptive immune response, mechanisms involved in LPS synthesis are still unknown. In this context we searched in B. bronchiseptica genome for putative glycosyltransferases. We found possible genes codifying for enzymes involved in sugar substitution of the LPS structure. We decided to analyse BB3394 to BB3400 genes, closed to a previously described LPS biosynthetic locus in B. pertussis. Particularly, conservation of BB3394 in sequenced B. bronchiseptica genomes suggests the importance of this gene for bacteria normal physiology. Deletion of BB3394 abolished resistance to naive serum as described for other LPS mutants. When purified LPS was analyzed, differences in the LPS core structure were found. Particularly, a GalNA branched sugar substitution in the core was absent in the LPS obtained from BB3394 deletion mutant. Absence of GalNA in core LPS alters immune response in vivo but is able to induce protective response against B. bronchiseptica infection.