Use of Fecal Streptococci as Indicators of Pollution in Soil

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1978, p. 711-717 0099-2240/78/0035-0711$02.00/0 Copyright i 1978 American Society for Microbiology

Vol. 35, No. 4

Printed in U.S.A.

Use of Fecal Streptococci as Indicators of Pollution in Soil H. J. KIBBEY, C. HAGEDORN,* AND E. L. McCOY Department of Microbiology, Oregon State University, Corvallis, Oregon 97331

Received for publication 28 November 1977

The survival, recovery, and identification of Streptococcus isolates from soil investigated by (i) examination of survival in soil under different moisture and temperature conditions, (ii) evaluation of media combinations for recovering fecal streptococci from soil, and (iii) partial identification of isolates from diverse habitats. Cool, moist conditions prolonged the survival of Streptococcus faecalis in soil for at least 12 weeks, whereas freezing was lethal, with the populations being reduced up to 95% when several freeze-thaw treatments occurred. Media evaluations indicated that both the efficiency of recovery and enumeration of the fecal streptococci from soil can be influenced by the combination of media used. Taxonomic data revealed a need to develop procedures to differentiate between isolates of fecal origin and plant-derived streptococci that possess many of the cultural reactions of S. faecalis. It was found that recent fecal isolates exhibited a much greater incidence of multiple antibiotic resistance than soil or vegetation isolates, and this characteristic, coupled with the use of enterococci as indicators of fecal contamination in soil systems, is discussed.

was

With the recent introduction of many new regulations at both the federal and state level controlling air and water pollution, the soil is being used more extensively as a means of disposing of human, livestock, and industrial wastes. Evidence is scarce on the consequences of using soil over a prolonged period of time as a sink for disposing of such wastes, even though this disposal, especially with sewage sludge and treated effluent, has increased dramatically over the last several years in both urban and rural areas. The greatest concerns in rural areas where waste disposal on soils is practiced are the maintenance of drinking water quality in rural watersheds and the potential for fecal organisms to contaminate crops grown on the same soils. In this respect, it becomes essential to monitor waste-amended soils so that potential hazards can be identified and evaluated in making decisions on the proper use of soil as a waste disposal system. The fecal streptococci have been used extensively as indicator bacteria in aquatic systems (6), and this precedent has also been used to monitor the level of fecal contamination in soil (3,9). Any organism used in such a manner must represent a fecal source, be foreign to the soil environment, and possess characteristics which allow its differentiation from any other closely related organisms. Such criteria have been applied to certain fecal streptococcal biotypes as indicators of soil pollution, although evidence indicates that plant-associated streptococci of nonfecal origin exist which closely resemble the

fecal streptococci (10, 12, 14). In addition to taxonomic confusion, there is uncertainty concerning the most suitable media to employ to recover fecal streptococci from soil, as well as which of the various biotypes of Streptococcus faecalis are of greatest sanitary significance. This paper examines the survival and isolation of fecal streptococci from contminated soil, presents data on S. faecalis biotypes from different habitats, and discusses the use of these organisms as indicators of fecal contamination in soil systems.

MATERIALS AND METHODS Streptococcus survival studies. The organism used was S. faecalis isolated from raw sewage from the Corvallis, Ore., Wastewater Treatment Plant. This strain did not hydrolyze starch, produced a reduced, hard, acidic curd from litmus milk, and was identified according to Sherman's criteria in Bergey's Manual (4). A spontaneous mutant of this strain was found which exhibited resistance to 80 gtg of streptomycin per ml. This biotype was used in the soil survival studies because the antibiotic resistance allowed differentiation from other streptococci which were already present in some of the soil samples. The antibiotic-resistant strain was grown in Difco brain heart infusion broth plus streptomycin for 48 h at 35°C, after which the cells from each liter of broth were pelieted by centrifugation (12,000 x g, 15 min), resuspended in 20 ml of phosphate buffer (pH 7.0), and stored at 40C. Five soil series (Table 1) were selected for survival experiments. All five are moderately extensive in westcentral Oregon, and the procedures used in determining the soil characteristics have been described (7).

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KIBBEY, HAGEDORN, AND McCOY

APPL. ENVIRON. MICROBIOL.

TABLE 1. Characteristics of the A-horizons for soils used in S. faecalis survival studies % Clay % Base saturation Soil pH % Organic matter Texture 87.4 22.7 6.8 6.0 Silt loam Dayton 76.7 36.1 4.3 6.6 Silty clay loam Dixonville 61.5 4.9 27.4 6.0 Silt loam Hazelair 42.6 47.0 5.2 5.6 Silty clay loam Jory 19.2 4.4 35.6 4.5 Gravelly loam Marty Soil series

Soil samples were collected from the A-horizons (upper 15 cm) with sterile trowels and placed in plastic pots 25 cm wide by 45 cm tall. The soil was added to each pot in approximately 7-cm increments, and a 20ml culture portion was sprinkled over the top of each soil increment. This procedure was repeated until the pots were filled and each contained a total of six 20-ml culture increments (representing 6.0 liters of centrifu-

gation-concentrated culture). Eighty pots were filled in this manner, 16 from each of the five soil series. Pots were incubated at 4, 10, 25, and 37°C under moisture pressure tensions of 0.0 bars (soil saturation), 0.3 bars (field capacity), 7.5 bars (50% field capacity), and 30.0 bars (air dried). The saturated samples were maintained by placing the pots in plastic buckets and adding water to the buckets periodically until the water level equilibrated at the rim of the plastic pots. The field capacity and 50% field capacity samples were weighed, the weight of the pot was subtracted, and the sterile water was added periodically to maintain the appropriate moisture status. The air-dried samples were allowed to dry before the soil was added to the pots, and no water was added except during the addition of the cultures. Core samples were withdrawn at 2-day intervals for the first 10 days of incubation and at 10-day periods thereafter for 120 days Cores were removed from the entire depth of each pot with a sterile 1.5-cm core auger, and each core was weighed and suspended in 9x its weight of sterile 0.5% peptone buffer (7). After the cores were withdrawn, the holes were filled with fresh, uninoculated soil. Following agitation in a Waring blender for 3 min at low speed, serial dilutions were spread over petri plates (in triplicate) containing Difco m-enterococcus (ME) agar plus 80 ,ug of streptomycin per ml. Colonies were counted after incubation at 35°C for 48 h. Twelve additional pots containing the Hazelair series were prepared, sampled, and assayed as described. These were maintained at the same four moisture levels (three pots at each). One pot from each level was subjected to the various freeze-thaw procedures. In treatment A, the pots were frozen for 8 weeks and then thawed; in treatment B, the pots were frozen and thawed biweekly for 8 weeks (four freezes); and in treatment C, the pots were frozen and thawed weekly for 8 weeks (eight freezes). All freezing was at -10°C; the thawing temperature was 25°C. The pots were assayed for S. faecalis populations weekly for 4 weeks after termination of the treatments. Media evaluations. One hundred soil samples from locations throughout Oregon were obtained from the Oregon State University Soil Testing Laboratory. These samples were largely from soils under agricultural use. Portions were aseptically removed from mail-in soil bags and stored at 4°C. Samples obtained

in this manner varied in moisture content (saturated to air dry) and length of storage time. One-tenth of a gram of soil was added to 10 ml of the following Difco media: azide dextrose (AD) broth, ethyl violet azide (EVA) broth, SF broth, KF streptococcus (KF) broth, ME broth, and enterococcus presumptive (EP) broth. Incubation was at 35°C for 48 h for all except EP broth, which was incubated at 45°C for 48 h. After incubation, all positive tubes were confirmed on ME and KF agars and on EVA broth. All six media were also evaluated by using artificially inoculated soil. Two pots containing the Jory soil series were inoculated as described above and incubated at 10°C under saturated moisture conditions. Serial dilutions of the core samples were used to inoculate 15-tube series (5 tubes by 3 dilutions) of the various media following procedures in Standard Methods (1). EP broth was incubated at 45°C, and the other media were incubated at 35°C for 48 h. Positive presumptive tubes were confirmed by streaking on ME agar.

Taxonomic examinations. Seventy-five isolates were classified according to the criteria in Bergey's Manual (4), the enterococcus identification key of Geldreich and Kenner (6), and the plant streptococcus groups designated by Mundt (13). Bergey's Manual criteria are comprised of those of Sherman (15) plus additional tests which include hemolysin production, sorbitol and arabinose fermentation, gelatin liquification, hydrolysis of hippurate, arginine deamination, reaction in litmus milk, methylene blue reduction, growth on bile-esculin agar, starch hydrolysis, growth at 45 and/or 10°C, and tolerances to 60°C, to pH 9.6, to 40% bile, and to 6.5% NaCl. The tests used by Geldrich and Kenner (6) include lactose fermentation, catalase, starch hydrolysis, reaction in litmus milk, tolerance to 6.5% NaCl, growth at 45 and/or 10°C, and growth in 40% bile broth. The sevenplant streptococcus types described by Mundt (13) are differentiated by first separating the known streptococcal species by using Sherman's criteria and then applying a combination of tests to atypical isolates. Twenty-five of the cultures were randomly chosen from the soil isolates, 25 were isolated from sewage sludge and influent samples obtained from the Corvallis Wastewater Treatment Plant, and 25 were recovered from plant samples. The sewage and plant isolations were performed on ME broth and confirmed on ME agar, and the plant samples consisted of a variety of types of live vegetation collected from undisturbed regions within the McDonald State Forest in western Oregon. American Type Culture Collection (ATCC) reference strains of S. faecalis (ATCC 19433), S. faecalis var. liquifaciens ATCC 27332), and S. bovis (ATCC 9809) were included with the unknown isolates.

VOL. 35, 1978

FECAL STREPTOCOCCI AS INDICATORS OF POLLUTION

Antibiotic sensitivity patterns. Sensitivities were determined by the disk diffusion technique by using high-concentration disks (2). Several well-isolated colonies were aseptically transferred from brain heart infusion agar plates to 4 ml of tryptose phosphate broth. Cultures were incubated for 2 to 5 h at 35°C after which their optical turbidity was adjusted with 0.85% saline solution to match the BaCl2 turbidity standard. Mueller-Hinton agar plates (BBL) containing 5.0% defibrinated sheep blood were inoculated in three directions with sterile cotton swabs dipped into the broth suspensions. The following antibiotic disks (BBL) were then pressed onto the plates: ampicillin (10 ug), bacitracin (10 U), cephalothin (30 tg), erythromycin (15 ILg), gentamicin (10,ug), novobiocin (30 ,tg), penicillin (10 U), and tetracycline (30 Ig). After 16 to 18 h at 35°C, zone diameters were measured, and isolates were assigned as resistant or not resistant. The three streptococcal ATCC strains were included, as well as Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923).

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TABLE 2. Average 95% population reduction times for S. faecalis amended to soil samples incubated under different moisture and temperature regimes Sol moisture equivalent

Soil water suction

Saturation Field capacity 50% Field ca-

0.0 0.3 7.5

Time required (days) for 95% reduction in at: no. of bacteria

(bars)

40C 94 60

10°C 250C 370C 80 53 29 43 38 16

35

29

22

8

23

18

9

5

pacity Air dried

30.0

7.0

RESULTS The soils used in the survival studies were chosen to represent a range of textural and chemical characteristics (Table 1). The data on the S. faecalis populations (Table 2) indicated that, regardless of the soil examined, the bacteria died more rapidly under the drier conditions at the warmer temperatures and survived the longest under moist conditions (soil saturation) at cooler temperatures (4 and 10°C). This trend was consistent for all soils, even though there were wide variations in the population reduction times between the five soil series (Fig. 1A and 1B). The average death curves of the bacteria in the different soils under saturation (Fig. 1A) and 50% field capacity (Fig. 1B) at 25°C are presented to illustrate that the populations declined at different rates in the various soils maintained at different moisture levels and that the populations declined less rapidly under saturated soil conditions regardless of the temperature of incubation. The average 95% reduction time for the bacteria under the saturated conditions (Fig. 1A) was 53 days and did not include the Dixonville series, as 95% of the streptococci in that soil had not expired by the termination of sampling at 120 days. The results of the freeze-thaw experiments demonstrated that the S. faecalis numbers were greatly reduced by all three treatments, and the numbers of viable survivors were the lowest in the treatment containing the most freeze-thaw intervals (Table 3). When the populations from all treatments were measured immediately after the final thaw, the number of survivors was greatest in the pots maintained at lower moisture levels. However, the opposite effect was observed after the 4-week assay period in that the survivors died off most rapidly in the pots

-j

0

Cl)

E 0) -j

N

uJ 0

0 -j

TIME (days) FIG. 1. Survival of S. faecalis in different soil series incubated at 25°C under moisture conditions of saturation (A) and 50% field capacity (B).

kept at the lower moisture levels after the final thaw and subsequent incubation of the treatments at 25°C. Exposing the S. faecalis populations to multiple freeze-thaw cycles resulted in the smallest numbers of survivors, and these survivors died off more rapidly than in the treatment where the cells had only been exposed to one prolonged freeze. The air-dried samples contained the largest numbers of survivors, because there was little ice formation during the treatments (and subsequent cellular damage by ice

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KIBBEY, HAGEDORN, AND McCOY

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TABLE 3. Effects of freeze-thaw treatments on S. faecalis populations in soil samples maintained at different moisture levels Population treatTreat- before(no. mna cells/g ofof soil)

ment'-ment

Soil moisture status

Saturation iII

Field capacity I

50% Field capacity

II

I

Air dried

II

I

II

2.4 x 106 3.8 x 104 2.8 x 103 4.7 x 104 1.9 X 102 7.4 x 104 2.1 x 102 4.3 x 105