Mycobacteria in boreal coniferous forest soils

Mycobacteria in boreal coniferous forest soils

FEMS Microbiology Ecology 23 (1997) 325^332 Mycobacteria in boreal coniferous forest soils Eila K. Iivanainen a *, Pertti J. Martikainen a , Marja Li...

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FEMS Microbiology Ecology 23 (1997) 325^332

Mycobacteria in boreal coniferous forest soils Eila K. Iivanainen a *, Pertti J. Martikainen a , Marja Liisa Raëisaënen b , Marja-Leena Katila c ;

a

Laboratory of Environmental Microbiology, National Public Health Institute, P.O. Box 95, FIN-70701 Kuopio, Finland

b c

Geochemical Department, Geological Survey of Finland, P.O. Box 1237, FIN-70211 Kuopio, Finland

Department of Clinical Microbiology, Kuopio University Hospital, P.O. Box 1777, FIN-70211 Kuopio, Finland

Received 25 November 1996; revised 21 May 1997; accepted 22 May 1997

Abstract

The occurrence of mycobacteria was studied in organic horizons of coniferous forest soils in Finland and related to environmental variables, i.e. plate counts of other heterotrophic bacteria, microbial respiration rate, chemical soil characteristics, vegetational characteristics and climatic conditions in the study period. Mycobacteria were isolated from all samples (n=47), with plate counts varying from 4.5 104 to 1.2 106 cfu g31 dry soil. The plate counts of mycobacteria correlated positively with those of other heterotrophic bacteria, microbial respiration rate and the contents of Ca and Mn. In factor analysis, the viable counts of mycobacteria and other heterotrophic bacteria, and respiration rate were grouped in the same factor emphasizing that mycobacteria and other heterotrophic bacteria had similar associations with environmental characteristics. The plate counts of mycobacteria and other heterotrophic bacteria and microbial respiration rate were similar in organic horizons of pine and spruce dominated forests. The large number of mycobacteria in all organic horizons indicates that boreal coniferous forest soils are important sources for these bacteria.

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Keywords :

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Atypical mycobacterium; Heterotrophic bacterium ; Coniferous forest; Vegetation; Soil

1. Introduction

The increasing importance of atypical mycobacteria as human pathogens [1^3] and the lack of evidence for human-to-human transmission of infections caused by these bacteria [3] have stimulated the research on their environmental occurrence and ecology. Several studies on natural waters, water distribution systems, and soils have shown the ubiquitous presence of these heterotrophs in the environ* Corresponding author. Tel.: +358 (17) 201375; Fax: +358 (17) 201155; E-mail: [email protected]

ment [4^6]. Some studies on the ecology of mycobacteria have suggested that low pH and high content of organic matter favor the occurrence of mycobacteria in the environment [7^10]. In general, however, there is little knowledge on factors a¡ecting the occurrence of mycobacteria in soils. Coniferous forests are the most common forest type in boreal regions. They have a podzolized soil pro¢le with an acidic organic horizon, which is the most important layer for microbial growth in these soils [11,12]. The chemical composition and microbiological characteristics of the organic horizon are highly dependent on litter production of vegetation. The boreal conifer-

0168-6496 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 1 6 8 - 6 4 9 6 ( 9 7 ) 0 0 0 4 0 - 8

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ous forests vary in their fertility and vegetation cover, and may then also vary in soil microbiological characteristics. In this study, we examined the occurrence of mycobacteria in relation to other heterotrophic bacteria in coniferous forest soils with di¡erent chemical and vegetational characteristics in nonpolluted areas in Finland.

sampling points in each experimental plot in June^ August 1990. For chemical analyses, two subsamples from di¡erent points were combined, air dried and stored at room temperature. For microbiological analyses, the remaining two subsamples were combined, sieved (mesh size 3 mm) and then stored at 320³C until they were processed. 2.3. Climatic data

2. Materials and methods

2.1. Study area

The forests were on a 550 km long linear belt crossing Finland at 63³ northern latitude (Fig. 1). The characteristics of the belt have been described in details earlier [9]. In this belt, each experimental plot (diameter 10^15 m) was located in the most common forest type of the representative drainage area (total 47 forest sites). The age of the tree stand was 60^100 years, and the dominant species were spruce (Picea abies) (69% of the areas) and pine (Pinus sylvestris) (31% of the areas). Birch (Betula pendula/Betula pubescens) grew as a minor species in 30% of the forest sites. The forests were typed according to the Finnish classi¢cation [13] as Oxalis-Myrtillus (OMT, 28%), Myrtillus (MT, 55%) or Vaccinium (VT, 17%) site types, listed in order of decreasing fertility. The soils were podzolized and had organic horizons of 2^10 cm. The sites were situated 10^190 m above the sea level (mean 120 m). 2.2. Sampling

Four subsamples of 1 l were collected from two

The local climatic data were obtained from the Finnish Meteorological Institute. The temperature data recorded was the mean air temperature during the 7 days before sampling. The precipitation data included the cumulative precipitation for the 3 days, and 1 and 2 weeks preceding each sampling. 2.4. Chemical analyses

The pH of the air dried soils were measured in 0.025 mol l31 NH4 NO3 solution (2.5 g soil in 25 ml of solution) [14]. Total contents of C and H were determined by infrared detector and total content of N by thermal conductivity detector after pyrolysis of organic material in £ow of oxygen at 900³C (Leco CHN-600 carbon-hydrogen-nitrogen determinator, Leco Corp., St. Joseph, MO). Total content of S was determined by infrared detector after pyrolysis of samples in £ow of oxygen at 1370³C (Leco S-132 sulfur determinator, Leco Corp.). For analyzing mobile metals, the air dried samples were extracted in 1 M ammonium acetate bu¡ered at pH 4.5 [15]. Concentrations of Al, Ca, Co, Cu, Fe, K, Mg, Mn, Na, Ni, Pb and Zn were determined by atom absorption spectrometry £ame method (model 4000 appa-

Fig. 1. Location of the study belt with the 47 forest sites in Finland.

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ratus; Perkin Elmer Corp., Germany). Concentrations of As and Cd were analyzed by graphite oven atom absorption spectrometry (model 3030 apparatus; Perkin Elmer Corp.). CO2 produced in microbial respiration in soil was analyzed with a Horiba pir-2000 infrared analyzer (Ionics Inc., Watertown, MA). 2.5. Microbiological analyses

The frozen soils were thawed at 4³C overnight. The moisture content was determined by drying the samples at 105³C for 24 h. For microbiological cultures, bacteria were detached from soil particles by homogenizing the thawed soils at 8000 rev min31 (Oci Omni-mixer, Omni Corporation International, Inc., Waterbury, CT) in trypticase soy broth (BBL, Becton Dickinson Microbiology Systems, Cockeysville, MD) for 2 min. Heterotrophic bacteria were determined on Winogradsky's salt solution agar with 1 ml of nutrient stock solution per liter (WSA) [16] and R2A agar (Difco Laboratories, Detroit, MI) [17], by incubating the plates in the dark at room temperature for 6 weeks. The decontamination methods and growth media for mycobacterial cultures were chosen on grounds of the earlier comparative study [18]. The homogenized samples were preincubated at 37³C for 5 h before decontamination by NaOH (0.3 mol l31 ), malachite green (0.6 mg ml31 ) and cycloheximide (0.5 mg ml31 ). Dilutions of 1031 and 1032 were made of each suspension. 50 Wl were inoculated on two parallel egg media (pH 6.5) supplemented with cycloheximide (0.5 mg ml31 ) and glycerol, and two parallel egg media (pH 6.5) supplemented with cycloheximide (0.5 mg ml31 ) and pyruvate. Cultures for mycobacteria were incubated at 30³C for 6 months. The acid fastness of the isolated colonies was checked by Ziehl-Neelsen staining, and selected acid fast isolates [18] were veri¢ed as mycobacteria by gas liquid chromatography analysis of fatty acid and alcohol composition [19]. The plate 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 measured to char-

327

acterize total microbial activity in soil. 5 g of the thawed soils were placed in 100-ml £asks (three replicates). Moisture contents of the soils were adjusted to 60% of their maximum water holding capacity, and the £asks were stoppered with rubber septa. The soils were then incubated at 14³C for one week, aerated and reincubated for another week. The CO2 accumulated during the second week incubation was measured as described above. The results were expressed as CO2 -C produced per dry weight and per day. 2.6. Statistical analyses

Non-parametric tests (SPSS/PC+ for the PC/XT/ AT, SPSS Inc., Chicago, IL) were used because normal distributions of the variables could not be obtained by transformations. The e¡ect of vegetation on chemical and microbial variables were examined by Mann-Whitney U test and by Kruskal-Wallis one-way analyses of variance. Relationships between the microbial and chemical variables were studied by correlation and by factor analysis using ranked values of parameters. 3. Results

3.1. Climatic data and chemical characteristics of the soils

Air temperature during the week preceding sampling varied between 7 and 18³C. Both the 3-day (range 0^27 mm), 1-week (range 0^56 mm) and 2-week (1^77 mm) precipitation before sampling varied greatly between the sites studied. The soils were acidic, pH ranging from 3.0 to 4.0 (Table 1). The total concentrations of C were high resulting in high C:N ratios. There was 6^8-fold variation in the concentrations of inorganic nutrients Na, K, Mg and Ca, and even 100-fold variation in the concentrations of some metals (Table 1). The concentrations of Cu, Co and Ni remained below the detection limits in most samples, and these variables were excluded from the statistical analyses. Some soil chemical characteristics varied by forest and vegetation types (Table 1).

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Table 1 Geographical characteristics of the sites, and the physical, chemical and microbiological characteristics of their soils Variable Dominant tree Forest type a Spruce (n = 32) Pine (n = 15) P OMT (n = 13) MT (n = 26) VT (n = 8) Pb Total range Height from the sea level 120 150 * 120 120 160 10^190 (m) Moisture (%) 130 160 120 140 160 34^290 pH 3.4 3.3 3.3 3.4 3.3 3.0^4.0 Total C (%) 42 47 ** 33 45 46 ** 23^51 Total N (%) 1.2 1.1 1.2 1.3 1.1 * 0.7^1.7 C/N 32 39 *** 30 34 41 *** 26^46 H (%) 6.0 6.6 ** 4.8 6.5 6.6 ** 3.2^7.1 Total S (%) 0.16 0.15 0.14 0.17 0.15 0.1^0.2 Ca (mg kg31 ) 1500 2000 * 1400 2000 1900 480^3800 Mg (mg kg31 ) 260 270 220 270 250 100^550 Na (mg kg31 ) 27 29 23 28 30 14^87 K (mg kg31 ) 640 740 640 640 770 250^1900 Fe (mg kg31 ) 50 18 *** 62 28 21 * 7.0^770 110 150 50 160 100 5.0^540 Mn (mg kg31 ) Zn (mg kg31 ) 20 34 * 15 29 35 * 0^50 Al (mg kg31 ) 190 130 * 300 120 160 37^1500 Cd (mg kg31 ) 0.35 0.43 * 0.33 0.35 0.49 ** 0.2^0.8 Pb (mg kg31 ) 17 21 * 14 20 24 * 5.0^95 0.2 0.3 0.2 0.1 0.4 0^0.8 As (mg kg31 ) Respiration rate 140 150 120 150 180 22^350 (Wg CO2 -C g31 day31 ) Heterotrophic bacteria on 4.2U107 3.9U107 3.8U107 3.7U107 1.5U106 ^1.0U108 3.6U107 WSA (cfu g31 ) Heterotrophic bacteria on 2.9U107 4.1U107 3.5U107 3.2U107 2.9U107 9.6U106 ^1.4U108 R2A (cfu g31 )c Mycobacteria (cfu g31 ) 2.7U105 3.7U105 2.5U105 3.8U105 1.9U105 4.5U104 ^1.2U106 The sites were classi¢ed according to the dominant tree species and the forest site type classi¢cation [13]. The medians and the minimum and maximum values of the soil variables are expressed per dry soil. a Statistical signi¢cance between the two dominant trees: * = P 6 0.05, ** = P 6 0.01, *** = P 6 0.001. b Statistical signi¢cance between the three forest types: * = P 6 0.05, ** = P 6 0.01, ***= P 6 0.001. c n = 26, 13, 8, 24 and 7 in spruce, pine, OMT, MT and VT groups, respectively. 3.2. Relationships between microbial and chemical characteristics

The plate counts of mycobacteria ranged from 4.5U104 to 1.2U106 cfu g31 dry soil, and they were similar in the di¡erent vegetation types of spruce and pine dominated forests (Table 1). There were wide variations in both plate counts of heterotrophic bacteria and soil respiration rate between the sites studied (Table 1) but similarly to mycobacteria, neither of the variables were associated with the quality of ground vegetation nor main tree stand (Table 1). WSA (median 3.9U107 cfu l31 ) and R2A (median 3.2U107 cfu l31 ) gave similar counts for heterotrophic bacteria (P=0.87 in Wilcoxon test).

The viable counts of mycobacteria correlated positively with the contents of Ca and Mn (Table 2). There were also positive correlations between the plate counts of mycobacteria and other heterotrophic bacteria and respiration rate. Overall, the bacterial counts and soil respiration rate correlated similarly with the climatic and chemical variables (Table 2). In factor analysis, six di¡erent factors were formed from the 22 variables included (Table 3). Factor 1 consisted of Fe, Al, C, H and Ca. As indicated by their negative loadings, the contents of Fe and Al correlated negatively with the contents of Ca and total C. The N, S and Mg contents of soil correlated negatively with the height from the sea level (factor 2). The Ca content was associated

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Table 2 Correlation coe¤cients between the numbers of mycobacteria and other heterotrophic bacteria, microbial respiration rate, and the geographical, climatic, physical and chemical variables Variable Mycobacteria Heterotrophic bacteria (WSA) Respiration rate r

Pa

r

Height from the sea level 0.17 Air temperature 30.02 Precipitation (3 days) 30.15 Precipitation (1 week) 0.13 Precipitation (2 weeks) 0.05 Moisture 0.06 pH 0.12 total C 0.16 total N 0.12 C/N 0.10 H 0.15 total S 0.20 Ca 0.36 * Mg 30.04 Na 30.08 K 0.10 Fe 30.16 Mn 0.35 * Zn 30.10 Al 30.22 Cd 30.11 Pb 0.11 As 30.13 Respiration rate 0.32 * Heterotrophic bacteria (WSA) 0.35 * Heterotrophic bacteria (R2A)b 0.48 ** Mycobacteria a Statistical signi¢cance: * = P 6 0.05, ** = P 6 0.01, ***= P 6 0.001. b n = 39. In other cases, n = 47.

with the soil pH, as were also the contents of Mn, K and Zn (factor 3). Heavy metals correlated with each other (factor 4). Air temperature correlated negatively with soil moisture, as shown by factor 5. The microbial variables, i.e. the viable counts of mycobacteria and other heterotrophic bacteria and respiration rate, were closely related to each other (factor 6). 4. Discussion

Our data showed an abundant occurrence of mycobacteria in boreal forest soils; they were isolated from all samples with the colony counts varying from 4.5U104 to 1.2U106 cfu g31 dry soil. In earlier

30.04 30.04 30.26 30.10 30.00 0.15 0.09 0.28 0.12 0.29 0.28 0.17 0.32 0.07 0.03 0.21 30.31 0.25 0.21 30.28 0.11 0.21 30.01 0.42 0.54 0.35

Pa

* * *

** *** *

Pa

r

0.05 30.30 30.25 0.16 0.21 0.34 0.24 0.39 0.30 0.34 0.42 0.38 0.59 0.32 0.08 0.61 30.09 0.50 0.42 30.25 0.44 0.43 0.04 0.42 0.34 0.32

* * ** * * ** ** *** * *** *** ** ** ** ** * *

studies, mycobacteria have been recovered from 70^ 92% of pasture, 86^97% of arable [20,21] and 84^ 100% of other types of soils [10,22,23]. Isolation frequencies of mycobacteria in forest soils have been low [20,21,24], e.g. in German forest soils frequencies have varied between 21% and 40% of the samples analyzed [20,21]. The di¡erence in isolation frequencies between the present and German study may be due to di¡erent growth media (egg media and acidi¢ed egg media in the German and the present study, respectively) or methods used (1 mol l31 NaOH followed by 50 mg ml31 of oxalic acid in the German studies in contrast to 0.3 mol l31 NaOH+0.6 mg ml31 of malachite green+0.05 mg ml31 of cycloheximide in the present study), or due to di¡erences in soil conditions between Finland and Germany. No

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Table 3 Rotated factor matrix of the soil and climatic variables Variable

Factors 1

3 3

Fe Al C

2

3

4

5

6

0.91 0.86 0.70

H

0.68

Ca

0.50

0.50

N

0.82

S

0.80

Mg

0.74

3

Height from the sea level

0.67

0.51

pH

0.88

Mn

0.86

K

0.56

Zn

0.50

0.57

Cd

0.77

As

0.68

Pb

0.67

Air temperature Moisture

3

0.80 0.72

Mycobacteria

0.80

Heterotrophic bacteria (WSA)

0.76

Respiration rate

0.56

Loadings above or equal to 0.50 in absolute values are shown.

chemical data was presented in the German reports.

been detected between water pH and mycobacteria,

Comparative data on total mycobacterial counts in

but not between water pH and other heterotrophic

di¡erent soils is very limited. Colony counts are only

bacteria [9]. In the soils examined here, the counts of

Mycobacterium avium, M. scrofulaceum (MAIS)

mycobacteria did not correlate with pH, as did not

[8,10]. In those studies, the maximum colony counts 1 dry soil [8] and 350 of MAIS have been 40 cfu g 1 dry soil [10]. The total viable counts of mycfu g

bial respiration. A likely explanation for these con-

cobacteria in our study were remarkably higher.

soils and waters studied earlier [8^10].

available

from

studies

M. intracellulare,

and

on

3

3

the counts of other heterotrophic bacteria or micro-

tradictory results is the very narrow pH range (3.0^ 4.0) in the present forest soils compared with the

Identi¢cation of mycobacteria in the present study

The results indicate that other factors than pH

was based on acid fast staining and analysis of fatty

were associated with the occurrence of mycobacteria

acid and alcohol composition of selected isolates.

in the present soils. The plate counts of mycobacteria

According to earlier studies, the numbers of hetero-

correlated positively with the contents of Ca and

trophic bacteria in the organic horizons of Finnish 7 1 dry soil [25] forest soils have varied from 10 cfu g 9 1 dry soil [26], as determined on soil to 10 cfu g

Mn. The same was true also for microbial respira-

extract agar. The median of heterotrophic bacterial

lated positively also with K content. The viable

counts on WSA in the forest soils examined here was 7 1 dry soil. 3.9 10 cfu g

counts

3

3

U

3

tion, which measures the total activity of heterotrophic microbial £ora in soil. The respiration corre-

of

other

heterotrophic

bacteria

correlated

positively with the content of Ca. The factor analysis

The few reports available on mycobacterial ecol-

showed that the contents of these three elements

ogy in soils have shown a connection between large

correlated positively with soil pH, as could be ex-

numbers of mycobacteria and high soil acidity [8,10].

pected for Ca and K [27^29]. Further, the microbio-

In Finnish brook waters, a similar correlation has

logical variables in our soils correlated negatively

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331

with the contents of Fe and Al, which agrees with an

study

earlier report on acidi¢ed soils [30]. The contents of

August, did not reveal correlations between myco-

Fe and Al usually increase with increasing acidity

bacterial

and

The

counts of other heterotrophic bacteria did not either

same was also shown by the present factor analysis.

correlate with soil moisture but the general microbial

These results suggest that an increase in occurrence

activity measured as respiration rate increased with

of mycobacteria was connected with an increase in

increasing moisture.

with

decreasing

content

of

Ca

[28,31].

done

in

a

counts

rather

and

short

the

period,

climatic

i.e.

June^

variables.

The

base cations and a simultaneous decrease in the con-

We conclude that the factors which are generally

centrations of Fe and Al. The same was true for the

known to support growth of heterotrophic bacteria,

growth of heterotrophic microbes overall.

such as increase in base cations, also promote the

There are results on the association between the occurrence

of

mycobacteria

and

some

survival

of

mycobacteria

in

these

extremely

acidic

sphagnum

soils. Large numbers of mycobacteria showed that

[32,33] or other plant species [34,35]. Organic hori-

boreal forests are important reservoirs of these bac-

zon of forest soil contains plant-derived organic ma-

teria.

terial of di¡erent quality. The humic substances are formed in decomposition of aboveground and belowground plant litters. Organic horizon also con-

Acknowledgments

tains living roots. Rhizosphere with root-derived exsudates highly favor microbial growth. It would be

This study was supported by the Academy of Fin-

possible that the di¡erent vegetation in various forest

land and the Finnish Anti-Tuberculosis Association

types could have some impact on the occurrence of

Foundation. We thank P. Karakorpi and the chem-

mycobacteria in the organic horizon. The classi¢ca-

ical laboratory of Geological Survey of Finland for

tion scheme for forests in Finland takes account for

technical assistance, and L. Korhonen for revising

both the tree stand and understorey vegetation [13].

the English language of the text.

However, no association between the counts of mycobacteria and vegetation type was found. No association between the forest type and the plate counts

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