Interactions of saprophitic soil bacteria and listeria monocytogenes via gaseous metabolites

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Soil is a multiple-factor system with a variety of diverse species of microorganisms and relationships between them; the study of all the components of a microbial ecosystem is therefore complicated. The study of the mechanism of regulation of activity of soil microorganisms is one of the central problems of soil microbiology. The presence of microorganisms in a given natural zone is determined not only by the environmental conditions but also by the existence of control performed by other members of the biocenosis. Such a control is one of the causes of microbial associations being formed in natural ecosystems.

Soil microbiocenosis is one of the most complicated biological communities. Various interrelationships occur between bacteria in the process of their activity, including those at the metabolic level [2], [9]. The interactions between populations via metabolites [4], including gaseous substances [7], are of crucial importance for maintaining the stability of microbial communities and the control of their species composition and production capacity. Both the stimulating and inhibiting action of volatile compounds of microbial origin on bacterial growth has been noted [3], [8].

Considering the fact that volatile compounds produced by microorganisms are able to act as intra- and interspecies regulators of microbial communities [1], [11], we attempted to study the character of the interaction between saprophytic soil bacteria and L. monocytogenes by means of gaseous metabolites.

To study the interactions between soil bacteria and L. monocytogenes from established microbial associations of brown forest and brown podzolic soils (the south of the Far East of Russia), we isolated saprophytic bacteria. A total of 20 strains of microorganisms differing in their cultural and biochemical properties were isolated. They were assigned to the genera Agrobacterium, Acinetobacter, Aeromonas, Micrococcus, Pseudomonas, Flavobacterium, and Bacillus according to Bergey's Manual of Determinative Bacteriology by means of API (Analytical Pro?le Index) tests (BioMerieux, France).

The method proposed by L.S. Tirranen [10] was used for the assessment of the action of volatile biologically active substances. The reactions of the test cultures to the effect of volatile metabolites of the study cultures were assessed by the difference in the colony size of the test organisms in the experiment and control on the second or third day of incubation, when the growth of the test culture colonies in the control was well-pronounced. The dishes with the test cultures that were not subjected to the action of the volatile products of metabolism of the study organisms served as controls. The statistical data processing was carried out according to G.F. Lakin [1990]. The mean of the colony diameter and the mean error were considered. The assessment criterion was the normalized standard deviation value to which the real value of this criterion was compared for the 95% level of significance.

The results obtained enabled us to assess the degree of influence of the volatile metabolites of saprophytic bacteria on the growth of L. monocytogenes during their interaction. Among them, 42% were negative (the volatile metabolites of the study cultures inhibited the growth of the test cultures), 30% were positive (the volatile metabolites of the study cultures stimulated the growth of the test cultures), and the remaining results (28%) were neutral. The zero interactions observed in the experiments may be weak positive or negative effects (less than 20% of the control), which were not determined by the method of investigation used. soil microorganism metabolite

The experimental results presented in the table demonstrate that among the bacteria studied an interaction exists via gaseous metabolites. Most of the study cultures released inhibitory volatile substances, which had a negative effect on the growth of the test cultures. The stimulating, i.e., positive effect of the soil bacteria occurred less frequently.

All the study cultures exerted a selective, both inhibitory and stimulating, effect on the growth of the test cultures. Bacteria probably produce a range of volatile compounds, their spectrum of action varying from broad to narrower. Of all the strains of the study cultures, the bacteria of the genera Pseudomonas and Acinetobacter revealed the highest inhibitory activity in relation to the L. monocytogenes. The stimulating action of these bacteria was observed in no more than 28% of cases. The volatile metabolites of Aeromonas exhibited the greatest stimulating activity. The inhibitory action of these bacteria was observed in no more than 8% of cases. Hence, it may be suggested that it is Pseudomonas and Aeromonas that exert the most significant effect on the growth and development of L. monocytogenes in the soils studied.

It should be noted that the volatile metabolites of the Flavobacterium and Bacillus investigated in the experiment did not appreciably influence the growth of the L. monocytogenes.

The reaction of the test cultures to the volatile metabolites of the study microorganisms was diverse. Thus, in most variations of L. monocytogenes was showed a mainly negative reaction. No strains showing a surge of growth in most cases were revealed.

Analysis of the experimental data revealed direct correlation (except bacilli and flavobacteria, which showed a neutral effect) between the inhibiting and stimulating effect of the culture; the absence of stimulation correlated with the presence of inhibition.

It is known that the growth rate of bacteria in different media may be different. Bacterial metabolites accumulated on different media may differ both in their qualitative and quantitative composition. We therefore studied the influence on bacterial growth of the gaseous metabolites of the same cultures grown on nutrient agar and plant agar. These comparative studies did not reveal a substantial difference between the actions of the volatile metabolites of the cultures grown on different media (the difference is statistically in significant, с > 0.05).

Thus, the stimulating and inhibiting effect of the volatile metabolites of saprophytic soil bacteria on L. monocytogenes was noted. Some gaseous substances released by soil bacteria may serve as nutrients for L. monocytogenes. For example, D.G. Zvyagintsev [12] stated that certain components of volatile metabolites of microbial origin (acetaldehyde, ethanol) could act as nutrients for microorganisms. G.M. Larionov [6] demonstrated that long-term cultivation of Pseudomonas pseudomallei in soil extracts resulted after six months in the activation of the fermentative properties and the synthesis of an inhibitor of concomitant microflora. In the opinion of L.S. Tirranen [10], the interaction of microorganisms by means of their gaseous metabolites is a widespread phenomenon, which may play a certain ecological role in natural habitats.

The data obtained allow us to assert that, at the metabolic level, a diverse character of interspecies interrelationships is observed between the bacteria, directly influencing L. monocytogenes growth and preservation in soils. The volatile compounds produced by soil bacteria may act regulators of microbial communities. In this regard, the preservation the L. monocytogenes in soils may be regulated by the products of metabolism of saprophytic soil bacteria.

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