Microbiological Monitoring of Lake Baikal

Abstract

The microbial community is not only a natural component of the ecosystem of rivers, reservoirs, and lakes, but also one of the main indicators of their ecological situation. For the public water supply, water reservoirs are often used which are affected by regular or accidental contamination, which greatly influences the water quality. Microbial indicators, limiting values of which are set by the relevant regulatory documents, are defined both for the water supply and for drinking water resources. We provide an overview of the main regulatory documents used in the Russian Federation to assess the microbiological quality of water resources and of methods and results about monitoring of Lake Baikal. Lake Baikal is a well-known example of an oligotrophic deep-water lake which serves as a source of drinking water. The microbial communities of Lake Baikal are formed in the unique extreme environmental conditions determining their metabolism: a low nutrient content and low average annual temperature in the water column. The microbiological indicators for the pelagic zone of the lake are quite constant, as was shown by long-term observations. However, in these ecological conditions the possibility must be taken into account that the water contains microorganisms potentially hazardous to human health. Systematic annual results have been presented (since 2005) of the microbiological monitoring of coliform bacteria, thermotolerant coliforms (TTC), coliphages, as well as Pseudomonas, Clostridium and Enterococcus in Lake Baikal. It is shown that the spatial distribution of allochthonous organotrophic and opportunistic bacteria is associated with the local anthropogenic impact: settlements, deltas of the main tributaries or domestic wastewater discharge. In the deep layers of the pelagic zone of the lake, no groups of opportunistic bacteria were found. Moreover, as the water depth increased, changes were found in the structure of the microbial community. Oligotrophic and psychrotrophic microorganisms were predominant there, while the amount of organotrophic microorganisms did not exceed the background level. The widespread prevalence of uncultivable bacterial forms in the natural environment and the ability of pathogenic and opportunistic bacteria to move into an uncultivable state make them of sanitary significance. It has been demonstrated that new species of heterotrophic microorganisms can be cultivated when the culture media and cultivation conditions are adapted. The experiments show the possible mechanisms of survival for opportunistic bacteria. During the cultivation of Enterococcus faecalis in the Baikal water at a low temperature, cells moved into a viable, but uncultivable state and restored their ability to reproduce after the addition of the nutrient.

Appendix: Sample Preparation and Laboratory Methods

The determination of the total number of cultivable microorganisms (TNCM), total coliform bacteria (TCB), TTC, coliphages and spores of sulfite-reducing clostridia was carried out according to the methodological instructions MUK 4.2.1018-01 (2002).

Heterotrophic bacteria isolation from the water of Lake Baikal was performed in all seasons on the standard hydrological sections (Fig. 1). Water sampling was conducted using Nansen water samplers and sterile bottles. To account for the total psychrotrophic, heterotrophic and oligotrophic bacteria counts, cultivation were carried out on R2A (Sigma-Aldrich, USA), FPA/10 (g/L: nutrient broth—2.0, bacteriological agar—15.0) and PYA (g/L: peptone—1.0, yeast extract—1.0, bacteriological agar—15.0) media, respectively (Parfenova et al. 2006). Petri dishes with R2A medium were incubated at 4 °C, the others at 20–25 °C. Enumerations were made for heterotrophic and oligotrophic bacteria after 8–10 days of incubation, and after 12–14 days for psychrotrophic bacteria.

Morphological and biochemical studies were performed for all isolated strains using gram staining, and studying their morphological, physiological and biochemical characteristics. Further identification of the cultures was carried out using test systems MMTE-1 MMTE-2, a set of NIB indicators and test systems for the non-fermenting bacteria Lachema.

For total bacteria number (TBN) enumeration, epifluorescence microscopy was used. Water samples were filtered through 0.22 μm filters, stained with 4,6-diamino-2-phenylindole (DAPI) and visualized with the Olympus epifluorescent microscope (×100).

Analyses of bacteria of the genera Enterococcus and Pseudomonas were carried out by means of membrane filtration: a volume of water (50, 100 mL) was passed through a filter with a pore size of 0.45 μm, then plated on selective media. For enterococci detection, a selected nutrient agar was used containing 2,3,5-trifeniltetrazoliyhlorid and sodium azide (NaN₃), which inhibited the growth of gram-negative bacteria. Bacteria of the genus Enterococcus form red or pink colonies on this medium. Plates were incubated at 37 °C. For more accurate results, filters with grown colonies were transferred to a medium containing bile, esculin and ammonium iron(III) citrate. Plates were additionally incubated at 44 °C for 2 h. Fecal enterococci have the ability to hydrolyze esculin and formed black-colored colonies. The results of enumeration express the number of colony-forming units (CFU) per 100 mL of water.

The antibiotic resistance of the strains was studied using the disk diffusion method for 8 antibiotics: streptomycin, tetracycline, vancomycin, benzylpenicillin, ciprofloxacin, erythromycin, gentamicin, and rifampicin. Analysis was conducted according to three groups: sensitive, intermediate and resistant (MUK 4.2.1890-04 2004).

A study of the cell morphology was carried out using transmission (TEM) microscopy. For TEM, visualization cells were fixed by glutaraldehyde at a final concentration of 2.5 % (2 h), washed with a phosphate buffer (pH 7.0) and postfixed with 1 % osmium oxide in a sodium phosphate buffer with potassium ferricyanide (0.2 %) for 2 h. Dehydration was performed in ethanol series: 30, 40, 50, 70 and 96 %, each for 10 min, then twice for 30 min in anhydrous acetone. The dehydrated material was impregnated into a mixture of epoxy resin (Araldite 502 Kit SPI USA) and acetone at a ratio of 1:1, then transferred to a Beem^TM polypropylene capsule in a fresh mixture of epoxy resins with a catalyzer (250 μl of DMP-30 to 20 ml of resin) and polymerized at 37 °C (12 h) and at 60 °C (48 h). The thin sections were obtained on the microtome Ultracut R LEICA (Austria), and mounted on palladium grids. Samples were contrasted with lead citrate (Reynolds 1963) and washed in 0.02 M NaOH, and in distilled water. The thin sections were analyzed using a Leo 906E transmission electron microscope (Zeiss, Germany).

For the experiments on the survival of enterococci cells under extreme conditions, two strains of E. faecium were used. The first, E. faecium N8, was isolated in 2005 from the water near the Kultuk settlement, and the second, E. faecium S11, was isolated in 2006 from the water of the Selenga River delta. Laboratory experiments were conducted with enriched cultures. Cells were grown in a 50 mL flask containing 30 mL of liquid selective media BHI (Difco) at 37 °C for one day. After biomass growth, cells were centrifuged and washed with a saline buffer (0.9 % NaCl). Then, aliquots containing about 1.2 × 10⁷ cells were inoculated into flasks with 100 mL of the non-sterile Baikalian water and incubated at different temperatures: 4, 20, and 37 °C. The control flask contained no bacteria. Enumerations of bacteria were performed daily by sampling an aliquot of 1 ml of water, which was plated on selective esculin agar with azide and kanamycin (HiMedia, M510). Experiments were carried out in triplicate and cells were cultured overnight at 37 °C.