Dr Eila Iivanainen, Laboratory of Environmental Microbiology, National Public Health Institute, PO Box 95, FIN-70701 Kuopio, Finland.
The occurrence of mycobacteria was studied in aerobic brook sediments from 39 drainage areas in Finland. The culturable counts of mycobacteria were related to climatic conditions, characteristics of the drainage area, chemical characteristics of the sediment and water, culturable counts of other heterotrophic bacteria, and microbial respiration rate in the sediment. The counts of mycobacteria varied from 1·1 × 102 to 1·5 × 104 cfu g−1 dry weight of sediment. They correlated positively with the proportion of the drainage area consisting of peatland, total content of C, content of Pb, microbial respiration rate in the sediment, and chemical oxygen demand of the water. The correlations of the mycobacterial counts with pH of sediment and alkalinity of water were negative. The results of the present sediment study and of the forest soil study published earlier strongly suggest that an increase in acidity increases the counts of mycobacteria and decreases the counts and activity of other heterotrophic bacteria. Mycobacterial counts were more than 100 times higher (per dry weight) in forest soils with pH 3·5–4·3 than in sediments with pH 4·5–6·3.
Infections caused by environmental mycobacteria are becoming increasingly common (Falkinham 1996). Human-to-human transmission of these infections has not been detected. Thus, the environment, either natural or man-made, is regarded as the only source of these infections. The occurrence of mycobacteria in different waters and soils has been extensively studied (Kazda 1973a,b; Falkinham et al. 1980; George et al. 1980; Brooks et al. 1984; Kirschner et al. 1992; von Reyn et al. 1993; Donoghue et al. 1997) but so far, the factors affecting the occurrence of mycobacteria in the environment are not fully understood. Mycobacteria in the brook waters and soils of Finnish drainage areas, which are also the subject of the present study, were studied previously and it was found that high numbers of mycobacteria in brook waters correlated with low pH and high content of organic matter in water, and with a high proportion of peatland in the drainage area (Iivanainen et al. 1993). These phenomena have been detected earlier in other waters (Kazda 1973a,b; Kirschner et al. 1992). The numbers of mycobacteria were also large in the naturally acidic coniferous forest soils of the drainage areas (Iivanainen et al. 1997) and results of other studies suggest that low pH might favour the occurrence of mycobacteria in soil (Brooks et al. 1984; Kirschner et al. 1992).
Aerobic brook sediment is a more conservative and stable environment than brook water. It may accumulate chemical compounds and thus reveal, even better than running water, the characteristics of the drainage area. The sediment–water interface usually exhibits high microbial activity (Jones 1982), and bacterial abundancy can be several orders of magnitude higher in shallow sediments than in the overlying water (Capone & Kiene 1988). Mycobacteria have occasionally been isolated from stream, pond (Wolinsky & Rynearson 1968) and ditch mud (Ichiyama et al. 1988), and from sediment from stream beds (Donoghue et al. 1997), but the factors affecting the occurrence of mycobacteria in sediment are not known. In this study, the aims were to discover whether the occurrence of mycobacteria was associated with environmental factors in brook sediments, and to compare the associations with those detected earlier in brook waters and forest soils of the same drainage areas.
Materials and methods
The brooks selected were on a 550 km long linear belt crossing Finland at latitude 63°N. The 39 drainage areas chosen for the sampling were mainly covered by woodlands (20–90% of the land area) or peatlands (3–75% of the drainage area). The characteristics of the brooks and drainage areas have been described in detail earlier (Iivanainen et al. 1993, 1997).
The loose sediments (thickness 0·5–5 cm) were collected at the outlets of the drainage areas, where the brooks were 2–9 m wide and 0·1–2 m deep. The sediments were sampled from a length of about 50 m of the brook, using equipment designed for brook sediment sampling consisting of a screen cup with 1 mm pores below which hangs a bag with 0·06 mm pores (Räisänen et al. 1992). During sampling, the sedimented, light, organic-rich material was suspended and passed through the screen cup into the bag. The coarse material (>1 mm) in the cup was excluded, and the mud retained in the bag was poured rapidly into a pail. The light organic fraction was swilled back into the bag and the water was gently wrung out. The samples obtained in this manner had dry weights of 15–64%. The samples were collected in June–August 1990 and divided into two parts; the first was used for chemical and the second for microbiological analysis. For chemical analyses, the samples were dried at 60 °C, homogenized with a plastic cutter and sieved through a 2 mm plastic mesh sieve. For microbiological analyses, the samples were stored at −20 °C until they were processed.
The local climatic data at the time of sampling were obtained from the Finnish Meteorological Institute. The temperature data recorded included the mean air temperature during the 7 d before sampling. Cumulative precipitation data for the 3 d, 1 week and 2 weeks preceding the sampling were also recorded (Iivanainen et al. 1993).
The chemical oxygen demand (COD) of the water was determined in the laboratory and the pH, at the time of sampling as described earlier (Iivanainen et al. 1993). The pH of the sediment was measured in a sediment–water suspension (5 g fresh sediment in 5 ml deionized water) (Model 611, Orion Research). Total C content was determined using an infrared detecter, and total N content using a thermal conductivity detecter, after pyrolysis of organic material in a flow of oxygen at 1000 °C (Leco CHN-600 carbon-hydrogen-nitrogen determinator). Total content of S was determined using an infrared detecter after pyrolysis of samples in a flow of oxygen at 1370 °C (Leco S-132 sulphur determinator). For the analysis of metal contents, the dried samples were extracted with a microwave-assisted concentrated nitric acid digestion (Anonymous 1990). Concentrations of Al, As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, La, Li, Mg, Mn, Mo, Na, Ni, P, Pb, S, Sb, Sc, Si, Sr, Th, Ti, V, Y and Zn were determined using an ICP-AES technique (Jarrell-Ash ICAP AtomComb Series 800).
The CO2 produced in microbial respiration (see below) in sediment was analysed with a Horiba pir-2000 infrared analyser (Ionics).
The frozen sediments were thawed at 4 °C overnight. The culturable counts of mycobacteria and heterotrophic bacteria, and microbial respiration rate, were determined from these moist sediments. The gravimetric moisture content was determined by drying a part of the sediments at 105 °C for 24 h. The dry weights obtained in this way were used to calculate the microbial results per gram of dry weight.
To determine the culturable counts, bacteria were detached from the sediment material by homogenizing the sample (4 g undried sediment) at 8000 rev min−1 (Oci Omni-mixer, Omni Corporation International) in 40 ml trypticase soy broth (BBL, Becton Dickinson Microbiology Systems) for 2 min. The homogenate was left to stand for 2 min, then 1 ml was taken for the determination of heterotrophic bacterial counts, and 15 ml for the determination of mycobacterial counts. Heterotrophic bacteria were cultured on R2A agar (Difco) (Reasoner & Geldreich 1985) and on Winograndsky's salt solution agar supplemented with 1 ml l−1 nutrient stock solution (WSA) (Olsen & Bakken 1987). The plates were incubated in the dark at room temperature for 6 weeks.
The selection of decontamination methods and growth media for the mycobacterial cultures was based on a pilot study carried out to optimize the efficiency of the enumeration. Each homogenized sample (15 ml) was centrifuged at 500 g at 4 °C for 5 min (Sorvall RC-5B, E. I. du Pont de Nemours and Co.) to settle the large particles. A 5 ml volume of the supernatant fluid was transferred to another sterile centrifuge tube and pre-incubated at 37 °C for 5 h to induce the germination of contaminating spore-forming microbes. The sample was then decontaminated with 5 ml 2 mol l−1 NaOH for 20 min. After centrifugation (8600 g, 4 °C, 15 min), the sediment was treated with 10 ml 50 g l−1 oxalic acid for 20 min. The re-centrifuged sediment was washed with 30 ml sterile distilled water, centrifuged again, and suspended in 3 ml sterile distilled water. Of this suspension, a dilution of 10−1 was made in sterile distilled water. A 50 μl aliquot of the suspended sediment and 10−1 dilution were inoculated onto two egg media (pH 6·5) supplemented with cycloheximide (0·5 g l−1) and glycerol, and two egg media (pH 6·5) supplemented with cycloheximide (0·5 g l−1) and pyruvate (Katila et al. 1989; Katila & Mattila 1991). The cultures for mycobacteria were incubated at 30 °C for 6 months. The acid fastness of the isolated colonies was checked using Ziehl-Neelsen staining, and randomly selected acid-fast isolates were verified as mycobacteria by gas liquid chromatography analysis of fatty acid and alcohol composition (Torkko et al. 1998). The culturable count of mycobacteria in a sample was calculated as the weighted mean of mycobacterial colonies growing on the two media inoculated with the two dilutions used.
Microbial respiration rate was used to measure total microbial activity in sediment. Three, 5 g samples of the thawed sediment were placed in 100 ml flasks; 5 ml deionized water were added and the flasks were stoppered with rubber septa. The three replicate samples were shaken in a vertical shaker (100 rev min−1, Swip SM25, Edmund Bühler) at room temperature for 1 week, aerated, and then shaken for another week. The CO2 accumulated during the second week of incubation was measured as described above.
Combining the data on the sediment and coniferous forest soils
To evaluate the association of pH with the numbers of mycobacteria and other heterotrophic bacteria, and with microbial respiration rate, the results from the present sediment study were combined with those from earlier studies of the soils of the same drainage areas (Iivanainen et al. 1997). In this evaluation, soil pH values measured in water suspension (4·5 g undried soil in 30 ml deionized water) were used, as sediment pH values were also measured in water suspension. Soil respiration rates, originally determined at 14 °C (Iivanainen et al. 1997), were conversed to correspond to the respiration at 20 °C used for the sediments. This was based on a separate experiment in which CO2 production in five randomly selected soil samples was measured at both 20 and 14 °C as described earlier (Iivanainen et al. 1997). The respiration rates were 40% higher (s.e. 7%) at 20 °C than at 14 °C. The original respiration rates of soils were multiplied by a factor of 1·4 and these values were used to compare the sediments and soils.
Relationships between the microbial and chemical variables of sediments were studied by non-parametric Spearman correlation analysis (SPSS/PC + for the PC/XT/AT). The Wilcoxon matched-pairs signed-ranks test was used to compare the two groups of variables, i.e. water pH with sediment pH, and R2A counts with WSA counts. The association between pH and the culturable counts of mycobacteria and other heterotrophic bacteria, and microbial respiration rate, was analysed using the Mann–Whitney U-test.
Climatic data and chemical characteristics of the sediments
Air temperature during the week preceding sampling varied between 9 and 16 °C. The precipitation before sampling varied greatly among the 39 sites studied (Table 1). The pH of the sediment (median pH 5·2) was lower than that of the overlying water (median pH 5·7) (P < 0·001 in the Wilcoxon matched-pairs signed-ranks test). There were eight- and 19-fold variations in the total contents of N and C, respectively. Of the other elements, a variation greater than 10-fold was detected in the contents of S, P, Mn, Co and Ni (Table 1). As most values of As, B, Cd, Mo and Sb remained under their respective detection limits, these variables were omitted from statistical analysis. The total content of C correlated strongly with the total content of N (r= 0·94, P < 0·001). Also, the contents of S (r= 0·63, P < 0·001), P (r= 0·53, P < 0·01), Ca (r= 0·56, P < 0·001), Pb (r= 0·36, P < 0·05) and Sr (r= 0·55, P < 001) correlated with the total content of C.
Table 1. Climatic data of the study sites, and chemical characteristics of the 39 sediments
The element contents are expressed per dry weight.
Relationships between microbial and chemical characteristics in sediments
The counts of mycobacteria ranged from 1·1 × 102 to 1·5 × 104 cfu g−1 dry weight (Table 2) and there was no clear geographical pattern (Fig. 1). Most of the isolates required 1–4 months to form visible colonies in the primary isolation. Wide variations were detected both in the culturable counts of heterotrophic bacteria and in the soil respiration rates between the sites (Table 2). Counts for heterotrophic bacteria were similar on R2A (median 7·2 × 107 cfu g−1 dry weight) and WSA (median 2·8 × 107 cfu g−1 dry weight) media (P= 0·87 in the Wilcoxon test).
Table 2. Microbiological characteristics of the 39 brook sediments
The values are expressed per dry weight.
Respiration rate at 20 °C (μg CO2–C g−1 day−1)
Heterotrophic bacteria on WSA (cfu g−1)
2·6 × 106
1·5 × 108
2·8 × 107
Heterotrophic bacteria on R2A (cfu g−1)
3·6 × 106
3·5 × 108
7·2 × 107
Mycobacteria (cfu g−1)
1·1 × 102
1·5 × 104
2·3 × 103
The culturable counts of mycobacteria correlated positively with the total content of C, content of Pb, and the respiration rate of the sediments with the proportion of peatland in the drainage area, and with the COD value of the brook waters (Table 3). The culturable counts of mycobacteria correlated negatively with water and sediment pH, and with the alkalinity of the water (Table 3).
Table 3. Correlation coefficients between the culturable counts of mycobacteria and other heterotrophic bacteria in sediment, microbial respiration rate in sediment and climatic and chemical variables
Culturable counts of other heterotrophic bacteria correlated positively with the respiration rate of the sediments (Table 3), and both correlated positively with the total contents of C and N and the contents of P, Ca and Sr. In addition, the counts correlated positively with the Mn content, and the respiration rate with the contents of S, Na and Pb. The heterotrophic bacterial counts and soil respiration rate did not correlate with sediment pH or with the proportion of peatland in the drainage area (Table 3). The contents of Ba, La, Li, Sc, Si, Th, Ti, V and Y did not correlate with the culturable counts of mycobacteria or other heterotrophic bacteria, or with microbial respiration rate (data not shown).
Relationships between microbial characteristics and pH in sediments and soils
When the data concerning the sediments and coniferous forest soils collected from the same drainage areas (Iivanainen et al. 1997) were combined, the association between culturable counts of mycobacteria and pH was strongly negative (Fig. 2a,b). In contrast, the culturable counts of other heterotrophic bacteria and the respiration rate were associated positively with pH (Fig. 2c–f). The contrary relationships between pH and mycobacteria, and pH and the other heterotrophic microbes, were even more distinct when the results were computed on the carbon content basis (Fig. 2).
To our knowledge, there are only a few reports on the numbers of mycobacteria in sediments. The only quantitative study we found was by Ichiyama et al. (1988) from Japan in which the average culturable count of slowly growing mycobacteria in undried sediments was 8·7 × 103 cfu g−1. When our results were calculated in a comparable way, i.e. per weight of undried sediment, the median count was 8·7 × 102 cfu g−1. The samples of our study were stored at −20 °C before culture. However, for soils which are naturally frozen in winter, this has been proved to be a reliable method for preserving samples (Stenberg et al. 1998). The long-term storage of brook water concentrates at −75 °C has increased, rather than decreased, the culturable counts of mycobacteria (Iivanainen et al. 1995). This is probably because freezing and thawing breaks up microcolonies or releases the attached bacteria from particles. It is also known that as pure cultures, many slowly growing mycobacteria survive as well at −20 °C as at −70 °C (Kim & Kubica 1973; Allen 1986). Reasons other than storage may therefore explain our lower yields. These include the different isolation methods used (the Japanese study used 0·5 mol l−1 NaOH for 20 min for decontamination, and Ogawa egg media supplemented with antibiotics), or different environmental conditions of the sediments studied.
It was shown earlier that the occurrence of mycobacteria in brook water, the sediments of which were examined here, is associated with the characteristics of both the drainage area and the water (Iivanainen et al. 1993). The same was also true for mycobacteria in the sediments. The culturable counts of mycobacteria correlated positively with the proportion of peatland, total content of C, contents of some heavy metals and respiration rate in the sediment, and with the COD of the water. However, the correlations between the counts of mycobacteria and the pH of the sediment and alkalinity of water were negative.
Peatland is known to be an important growth environment for mycobacteria (Kazda 1977, 1978; Kazda et al. 1979). The presence of peatland is also one of the main factors associated with the occurrence of mycobacteria in water (Kirschner et al. 1992; Iivanainen et al. 1993). Mycobacteria in brook waters and brook sediments may have been leached from surrounding peatland. Moreover, the run-off from peatland carries organic matter which may enhance the survival and/or growth of mycobacteria in the sediment. The COD of the brook waters increases as the proportion of peatland and the precipitation increase (Iivanainen et al. 1993). The positive correlation between the COD and counts of mycobacteria in the sediments indicates that the organic matter from peatland provides an important carbon supply for mycobacteria. However, total content of carbon in the sediment did not correlate with the COD of the water or with the presence of peatland, indicating long-term accumulation and diagenesis of sedimented organic matter. The accumulated organic matter contains nutrients available for mycobacteria, as indicated by the positive correlation between the cfu of mycobacteria and the total content of carbon. Our results agree with the findings of Kirschner et al. (1992), who showed that the numbers of the Mycobacterium avium–Myco. intracellulare–Myco. scrofulaceum complex increase with increasing amount of humus material in the water.
It was found that R2A agar with a low nutrient content (originally developed for the enumeration of bacteria in potable waters (Reasoner & Geldreich 1985)) was as suitable as WSA agar (originally developed for soil bacteria (Olsen & Bakken 1987)) for the enumeration of heterotrophic bacteria in sediment. In contrast to the numbers of mycobacteria, the numbers of other heterotrophic bacteria and the respiration rate of the sediments did not correlate with the proportion of peatland or the COD of the water. As with mycobacteria, however, the heterotrophic bacterial counts and the respiration rate correlated positively with the total C and N content of the sediment, i.e. with the accumulated organic matter of the sediments, as might be expected (Bott & Kaplan 1985; Sander & Kalff 1993). The culturable counts of mycobacteria and the respiration rate correlated similarly with the total content of C, which may explain the positive correlation between them. A similar correlation has also been detected in boreal forest soils (Iivanainen et al. 1997).
In the sediments, correlations were detected between some elements (i.e. S, P, Mn, Pb, Ca and Sr) and the culturable counts of either mycobacteria or other heterotrophic bacteria, or respiration rate. These elements may occur in the organic compounds (Kabata-Pendias & Pendias 1992; Kullberg et al. 1993; Brady & Weill 1996) and in our sediments, they correlated positively with the total content of C, as was also observed in a large geochemical survey of Finnish brook sediments (Lahermo et al. 1996). The correlation of these elements with organic matter was probably the main reason for their positive correlation with the culturable heterotrophic counts and/or microbial respiration.
The culturable counts of mycobacteria in the sediments correlated with low pH and low alkalinity of the brook waters. The negative correlation with pH has been demonstrated before in water (Kazda 1973a; Kirschner et al. 1992; Iivanainen et al. 1993), floodplain soils (Brooks et al. 1984) and soils associated with swamps (Kirschner et al. 1992). When our sediment data were combined with our earlier data concerning coniferous forest soils from the same sampling sites, allowing examination over a wide pH range (3·5–6·3), it was observed that mycobacteria were significantly more common in acidic environments. The average mycobacterial counts (per dry weight) in the coniferous forest soils (pH range 3·5–4·3) (Iivanainen et al. 1997) were more than 100 times higher than those in the sediments (pH range 4·5–6·3). The decontamination method used for the soils was milder than that needed for the sediments, but this can only explain a minor part of the great difference in the counts (Iivanainen 1995). Mycobacteria differed distinctly from other heterotrophic bacteria in their relationship with pH. Both the culturable counts of heterotrophic bacteria and the respiration rate were higher in the sediments, and correlated highly positively with pH in the combined soil and sediment material. The positive correlation with pH had been detected earlier for respiration rate in forest soils (Martikainen et al. 1989; Smolander et al. 1994). Thus, the environmental characteristics of the sediment highly favoured the occurrence of heterotrophic microbes other than mycobacteria.
The culturable counts of mycobacteria and other heterotrophic bacteria, and the microbial respiration rate, are expressed here on both dry weight and carbon content bases. The latter takes into account the very different amounts of energy and carbon supply in the two environments (median total C content in sediments 7% and in soils 44%), which might influence the results (Sander & Kalff 1993; Raubuch & Beese 1995; Falih & Wainwright 1996). The analysis based on the C content also compensates for the effect of the difference in bulk density, which was higher in the sediments, due to their lower organic matter and higher mineral contents. In analyses based on the C content, the relationship of mycobacteria to pH was about the same as in analyses based on dry weight, but the positive correlations between the numbers of other heterotrophic bacteria and respiration rate and pH were stronger. These results show that the general conditions affecting the growth of heterotrophic microbes, e.g. substrate and O2 availabilities, were good in the sediments and confirm the importance of low pH as a selective factor promoting the growth and/or survival of mycobacteria. This is probably explained by the tolerance and, to some extent also, the preference, of mycobacteria for acidic conditions (Chapman & Bernard 1962; Portaels & Pattyn 1982; Katila et al. 1989; Katila & Mattila 1991), and by the simultaneous decrease of other heterotrophic microbes, which gives mycobacteria a competitive advantage. The fact that the contrary relationships between pH and mycobacteria, and pH and other heterotrophic microbes, were more pronounced when the results were compared on a total C rather than on a dry weight basis, shows that different organic matter contents should be taken into account when comparing the microbiological variables in different types of samples. The same conclusion has previously been reached for forest soils of different characteristics (Smolander & Mälkönen 1994).
In conclusion, the occurrence of mycobacteria in brook sediments correlated with a large proportion of peatland in the drainage area, high organic matter content and low pH, as has earlier been found in the waters of the same brooks (Iivanainen et al. 1993). In the combined sediment and soil material, the occurrence of mycobacteria was strongly negatively associated with pH. In this respect, mycobacteria differed clearly from other heterotrophic microbes which were favoured by an increase in pH. Our results concerning naturally acidic environments suggest that acidification of the environment may increase the occurrence of mycobacteria. The acidic environments in northern latitudes have a low content of buffering base cations, and at the present level of acid deposition, they are susceptible to further acidification (Sverdrup & Warfvinge 1988) and perhaps to further increases in the occurrence of mycobacteria. Compared with brook water, brook sediment is a more conservative environment, and this may influence the mycobacterial flora in the sediments. Species variation between the waters examined earlier (Katila et al. 1995) and the sediments will be investigated in future studies.
This study was financially supported by The Academy of Finland and The Finnish Anti-Tuberculosis Association Foundation. The authors thank P. Karakorpi and the chemical laboratory of the Geological Survey of Finland for technical assistance.