APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1978, p. 341-348 0099-2240/78/0036-0341$02.00/0 Copyright i 1978 American Society for Microbiology
Vol. 36, No. 2
Printed in U.S.A.
Fecal Coliform Elevated-Temperature Test: a Physiological Basis WILLIAM S. DOCKINS AND GORDON A. McFETERS* Department of Microbiology, Montana State University, Bozeman, Montana 59717 Received for publication 14 March 1978
The physiological basis of the Eijkman elevated-temperature test for differentiating fecal from nonfecal coliforms was investigated. Manometric studies indicated that the inhibitory effect upon growth and metabolism in a nonfecal coliform at 44.50C involved cellular components common to both aerobic and fermentative metabolism of lactose. Radioactive substrate incorporation experiments implicated cell membrane function as a principal focus for temperature sensitivity at 44.5°C. A temperature increase from 35 to 44.50C drastically reduced the rates of [14C]glucose uptake in nonfecal coliforms, whereas those of fecal coliforms were essentially unchanged. In addition, relatively low levels of nonfecal coliform ,B-galactosidase activity coupled with thermal inactivation ofthis enzyme at a comparatively low temperature may also inhibit growth and metabolism of nonfecal coliforms at the elevated temperature. Some waterborne coliforms not normally considered to be of fecal origin are able to ferment lactose at 44.50C (6, 12, 20), and under certain conditions these organisms may be present in relatively high numbers in water samples (2). These findings constitute a basis for controversy over the validity of the elevated-temperature test for indicating the potential presence of pathogens in the aquatic environment. The widespread use and sanitary implications of the elevated-temperature procedure in detecting fecal water pollution stress the importance of determining and understanding the physiological mechanisms involved in colifonn growth and metabolism at 44.50C. A knowledge of the biochemical basis would also be of value in the formulation of improved fecal coliforn assay procedures. The primary objectives of this investigation were to locate the cellular site(s) of temperature sensitivity of nonfecal coliforms which might account for their failure to ferment lactose or glucose at 44.50C and to describe the physiological characteristics of coliforms insofar as they relate to the elevated-temperature test. MATERIALS AND METHODS
The coliform group of bacteria has been defined as "all the aerobic and facultative anaerobic, gram-negative, nonsporeforming, rodshaped, bacteria that ferment lactose with gas formation within 48 hours at 35C" (1). This group has long been used as an indicator of fecal contamination of natural waters. Because waterborne coliforms can originate from sources other than the intestinal tract of warm-blooded animals, such as from the soil or the surface of vegetation and insects (7, 8), it is desirable to determine the source of these organisms before attempting to relate them to fecal contamination and the potential presence of pathogens. A number of different procedures have been devised to separate fecal from nonfecal coliforms; however, the most successful and widely accepted technique involves incubation of coliforms at elevated temperatures. The first elevated-temperature test proposed by Eijkman (5) differentiated fecal from nonfecal coliforms by the ability of fecal coliforms to ferment glucose in a glucose-peptone broth at 460C. The current and most widely used elevated-temperature procedure relies upon fecal coliform fermentation of lactose in a highly buffered medium, with the production of hydrogen and carbon dioxide at 44.50C. These improved procedures are largely the result of the early work done by Perry and Hajna (10, 11, 21) and more recently by Geldreich et al. (6, 7). These workers and Mishra et al. (20) have reported a high correlation of gas production from lactose at 44.50C with coliforms that are normally associated with fecal pollution.
Cultures. The enteric bacterial cultures used in these studies were obtained from the Montana State University (MSU) Culture Collection or the American Type Culture Collection or were isolated by membrane filtration from streams in the area of Bozeman, Mont. All organisms conformed to the coliform designation as defined by the American Public Health Association (1). Stream isolates were further differentiated by indole, methyl red, Voges-Proskauer, and citrate (IMViC) classification and by the ability of the orga341
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DOCKINS AND McFETERS
nism to ferment lactose at 44.5°C in EC broth (Difco Laboratories) with the production of gas. Those coliforms capable of fermenting lactose with gas production at 44.5°C were termed fecal coliforms (1) and included Escherichia coli B (MSU culture collection no. 164) and stream isolates of IMViC types ++-(five strains) and -+--. Coliforms unable to ferment lactose at the elevated temperature (nonfecal coliforms) included Klebsiella pneumoniae ATCC 13883 and stream isolates of IMViC types -+++, -+-+, and -+--. All cultures were maintained on nutrient agar (Difco) and stored at refrigeration temperatures. Manometric methods. Cells employed in experiments with EC medium were grown in tryptic soy broth (Difco)-0.3% yeast extract-0.25% glucose (TSY) at room temperature and were aerated by shaking. The coliforms were harvested after 18 h by centrifugation at 3,000 x g (Sorvall, RC2-B), washed twice, resuspended in cold standard phosphate buffer (1), and standardized to 0.60 absorbance at 660 nm (A60) with a Varian Tectron model 635 spectrophotometer. Cells used in experiments in which TSY broth was the medium used were grown without shaking at 35°C in TSY broth. These cells were harvested after 18 h, washed once, and resuspended in cold phosphate buffer to Ass of 0.75. Single side-arm flasks (Gilson) were used in all manometric experiments. In experiments measuring 02 uptake, 0.5 ml of 40% KOH and a fluted filter paper wick were inserted into the center wells of each flask. Aliquots (4 ml) of the specified medium were added to the main chamber, and 1 ml of a cell suspension was placed in the side arm of each flask. Control flasks underwent similar preparation except that 1 ml of phosphate buffer was added to the side arm in place of the cell suspension. The flasks were attached to a Gilson differential respirometer and allowed to equilibrate at 44.50C (±0.20C, measured with standard thermometer) for approximately 20 min. In experiments that were done anaerobically, the respirometer flasks were purged of 02 by shaking them for 15 min before the equilibration time as nitrogen gas was passed through them. The 1-ml cell suspension in the side arm was then tipped into the main chamber, and the manometer readings were taken at 10- or 15-mm intervals. [14Cjglucose uptake. Coliforms used in ['4C]glucose uptake experiments were grown without shaking at 350C in TSY broth. The cells were harvested after 12 h, washed once with phosphate buffer, and resuspended in TSY broth to an Ass of 0.1 to 0.2. Flasks containing 60 ml of the cell suspension in TSY broth were equilibrated at 35 or 44.50C in water baths. At zero time 0.5 ml of uniformly labeled ['4C]glucose (0.5 ,uCi/ml, New England Nuclear Corp.) was added to the suspension, and a sample was taken immediately and at timed intervals thereafter. Aliquots (3 ml) of the culture suspension were removed for biomass determination (Ass), and 10-ml aliquots were filtered through a 0.45-,um filter (Millipore Corp.) for cellular radioactive glucose uptake measurements. Filters were washed with distilled water to remove extracellular ['4C]glucose, dried for 15 min at 105°C, and placed in poly Q scintillation vials (Beckman Instrument Co.). Toluene (4 ml) and 9 ml of Aquasol (New England
APPL. ENVIRON. MICROBIOL. Nuclear) were added to each vial. Labeled carbon counts and external standards were measured for each vial with a liquid scintillation counter (Beckman LSC 100) set at 5% error. Background counts were determined by filtering 10 ml of TSY broth and preparing this sample in the same manner as described above. Because of the similarity of the external standard counts, data were directly compared and expressed as counts per minute per absorbance unit (cpm/A6w). The maximal rate of uptake during the first 40 min of incubation was graphically calculated by determining the slope of the steepest sustained portion of the curve (counts per minute per A6s versis minutes). Temperature shift. Coliform organisms were grown at 35°C with shaking in TSY broth (without glucose) containing sodium acetate at a concentration of 1.0%. Cells were harvested after 18 h, washed twice with cold phosphate buffer, and resuspended in fresh TSY broth (without glucose). The A6s of this suspension was adjusted to fall within the range of 0.1 to 0.2. The suspension was equally divided into two flasks which were placed into a 35°C water bath and allowed to equilibrate for approximately 20 min. Aliquots (10 ml) of 10% lactose were added to each flask. The Ass of each cell suspension was measured at 15-min intervals for 3 h. After 1 h one flask was removed from the 35°C bath and placed at 44.5°C while the other flask remained at 35°C. ,B-Galactosidase assay. Coliform organisms were grown in TSY broth (without glucose) to which lactose at a final concentration of 1.0% was added. These cultures, fully induced for ,B-galactosidase, were harvested after 18 h, washed once, and resuspended in cold phosphate buffer. The A6s of the cell suspensions was adjusted to 1.25, and the cells were broken with a Bronwill Biosonic IV sonic oscillator set at 90% of maximum intensity. Sonication time was 15 min and the temperature of the suspension was not allowed to exceed 20°C. This method of sonication resulted in approximately 98% cell death. The extracts were then centrifuged at 3,000 x g for 5 min to remove remaining cells and larger cell fragments. The total protein contained in each extract was determined by the method of Lowry et al. (17). All cell-free extracts were stored on ice until use. f-Galactosidase activity was measured in the sonic extracts by a modified o-nitrophenyl-,8-D-galactopyranoside (ONPG) hydrolysis method (15,23). A 2.0-ml aliquot of 1.0 mM ONPG was added to 5 ml of the extract, and the mixture was incubated at a prescribed temperature for exactly 10 or 20 min. At the end of the incubation period, 2.0 ml of 0.5 M NaCO3 was added, and A420 was measured. In all assays the ONPG used was equilibrated to the temperature of the assay before addition. Results are expressed as A420 per minute per milligram of protein, and each data point is the average of three values. Aldolase assays. The preparation procedure for sonic extracts used in the aldolase assays was the same as that used for the f,-galactosidase assays except that tris(hydroxymethyl)aminomethane buffer (0.05 M, Sigma Chemical Co.) was substituted for phosphate buffer. Aldolase assays were performed by a modification of the spectrophotometric method of Jagannathan et al. (13). Aliquots (2 ml) of hydrazine sulfate
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FECAL COLIFORM ELEVATED-TEMPERATURE TEST
(3.5 mM in 0.1 mM ethylenediaminetetraacetic acid) were placed into a cuvette and allowed to equilibrate to 35 or 44.5°C in a spectrophotometer equipped with a recorder and a jacketed cuvette holder attached to a circulating heater. Fructose 1,6-diphosphate (0.10 ml, 12 mM) was then added, and the change in Ass over a 3-min interval was measured and recorded. Data were expressed as the initial slope of the Ass versus time per milligram of protein in the sonic extract (Ams per minute per milligram of protein). Aldolase assays were performed in triplicate, and each data point presented is the average of three initial
slopes.
RESULTS The results of manometric experiments in EC broth utilizing both a fecal coliforrn isolate (IMViC ++--) and a nonfecal coliform isolate (IMViC-+++) are shown in Fig. 1 and 2. The fecal coliform was capable of using lactose under both fermentative and aerobic conditions at 44.50C. The nonfecal coliform did not evolve gas from lactose at 44.50C in the respirometer under anaerobic conditions and furthermore did not metabolize lactose aerobically, as evidenced by the lack of oxygen uptake. Respiration studies in TSY broth gave results similar to those in EC broth (Fig. 3). Coliforms capable of a positive reaction in EC broth at the elevated temperature (44.50C), including an IMViC type ++-- isolate and E. coli B, showed a high respiration rate in TSY broth at 44.50C, whereas two nonfecal isolates (IMViC-+-+ 200
and -+++) demonstrated a miniimal rate of respiration at that temperature. The nonfecal organisms, however, showed minimal respiration in TSY broth at 44.50C, in contrast to the lack Of 02 uptake that was observed in EC broth at that temperature. Labeled glucose uptake studies were done to examine the effect of the elevated temperature upon bacterial transport functions (Table 1). Both nonfecal and fecal coliform isolates were capable of incorporating [14C]glucose at 350C in TSY broth and the maximal rates of uptake (counts per minute per A6ws) at this temperature were similar for the organisms tested. At 44.50C the fecal coliform isolates transported [14C]glucose at a rate comparable to that observed at 350C. In sharp contrast to this, nonfecal cohlforms showed little uptake of the labeled glucose at 44.50C compared with the maximal uptake rate at 350C. The rates of glucose incorporation (counts per minute per A6w, per minute) shown in Table 1 are not directly comparable between each organism tested because different activities of labeled glucose were used in different experiments; however, the ratio of gluocose uptake at the two temperatures can be directly compared for the various coliforms examined. A shift in growth temperature from 35 to 44.50C was followed where a nonfecal coliform (IMViC, -+++) was growing in TSY broth containing lactose. This experiment demonstrated
v-* FECAL COLSFORW (IMViC++--) o-O NON-FECAL COLIFORM (IMVIC -+++)
50 w
a.
o
343
100
MINUTES
FIG. 1. Comparison of respiration rates for fecal and nonfecal coliforms in EC broth at 44.5°C.
344
DOCKINS AND McFETERS
APPL. ENVIRON. MICROBIOL.
250 FECAL COLIFORM (IMViC+ +-- ISOLATE) O--O NON-FECAL COLIFORM (IMVIC-+++ ISOLATE)
2001
150w 0 w
100
50
30
d'0
--9 0------Q--
MINUTES
0
o
180 0 -----o----° 6
240
210 eo- --a
-
-O
FIG. 2. Comparison of gas evolution rates (CO2 + H2) for fecal and nonfecal coliforms in EC broth at 44.50C. Respirometer flasks were gassed with 1 00% nitrogen for 15 min to attain fermentation conditions.
that the high temperature stopped active cell division (Fig. 4). The temperature of the shifted suspension equilibrated to 44.50C from 350C within 10 min. The first observable change in the growth rate occurred within 30 to 60 min after the shift, and active growth of the culture gradually ceased between 30 and 90 min. A second differential effect of the elevated temperature was observed which involved the enzyme f8-galactosidase of fecal and nonfecal coliforms. f8-Galactosidase assays were done at temperature increments of approximately 5°C throughout the range of 10 to 45°C. The levels of f1-galactosidase activity in sonic extracts of fully induced fecal coliform cultures were consistently higher than those seen in comparable preparations from nonfecal coliforms (Fig. 5). Optimal activity of this enzyme in freshly prepared sonic extracts of fecal coliforms typically occurred at 30 ± 2°C, and the activity decreased rapidly as the temperature increased above 35 to 380C. At 44.50C fecal coliform f3-galactosidase activity was 25 to 50% of the optimal activity; however, nonfecal coliform /3-galactosidase activity was generally low or not measurable at that temperature. f8-Galactosidase activity was measured over the 10 to 45°C temperature range
for two additional sonically disrupted cultures not shown in Fig. 5. An E. coli B culture was similar to the two fecal coliforms shown but was slightly lower than those organisms in the level of enzyme activity that was observed. A nonfecal K. pneumoniae culture showed little fl-galactosidase activity at any of the assay temperatures. Spectrophotometric assays for aldolase activity were performed on sonic extracts of fecal and nonfecal coliforms to study the effect of the elevated temperature on this enzyme and to determine whether the reaction kinetics were affected by increased temperatures in the same manner as fB-galactosidase. Results of the aldolase assays for fecal coliforms (IMViC ++-and E. coli B) and nonfecal coliforms (IMViC -+++ and -+-+ isolates) indicate little difference between the two types of coliforms (Table 2), and the activity of that enzyme increased with temperature between 35 and 44.5°C regardless of the coliform type. DISCUSSION Although the elevated-temperature test has been used successfully for many years and several studies have addressed the sanitary significance of the procedure, the physiological basis
FECAL COLIFORM ELEVATED-TEMPERATURE TEST
VOL. 36, 1978
345
35O *-*
FECAL COLIFORM (L.gj U) *-4 FECAL COLIFORM(IMVIC + +--) °-O NON-FECAL COLIFORM (IMVIC -+++) a-O NON-FECAL COLIFORM (IMVIC -+-+)
,b200 w
oID150 I
SO FJ l
I
0~~~~~~~~~~~~
l
~ 100 50
100~~~~~
30
90
so
1 20
1 50
MINUTES
FIG. 3. Comparison of respiration rates for fecal and nonfecal coliforms in TSY broth at 44.50C.
TABLE 1. Incorporation of '4C-labeled glucose by fecal and nonfecal coliforms in TSY broth at 35 and
44.50C cpm/A,, per % Uptake colOrganism IMViC Fecal iform
FC1 BR6 BR4 BR1 BR1O NF1 NF2960 T8
++-++-++-++-++--+++ -+-+
+ + + + + -
min at:
35°C 44.50C 52.0 63.0 38.3 32.5 46.7 42.9 60.0 46.6 76.7 110.0 92.5 18.5 85.0 8.9 65.7 8.8
at 44.50C/
% uptake at 35°C 1.21 0.85 0.92 0.78 1.43 0.20 0.10 0.13
underlying the test has received little attention. Hendricks (12) examined several Enterobacter species using manometric techniques and suggested that the effects of the elevated temperature are focused upon the formate dehydrogenase complex. He postulated that there are at least two distinct biochemical types of Enterobacter in the aquatic environment and that the results of the elevated-temperature test de-
pended upon whether or not a coliform possessed formate dehyrogenase activity at 44.50C. The manometric data and growth curves obtained in this study indicate that the fecal coliform phenotype is a manifestation of more than a temperature effect upon the activity of the formate dehydrogenase complex in coliform bacteria. Although the elevated-temperature test is defined in terms of fermentative gas production from lactose at 44.50C, results of this study indicated that aerobic metabolism of lactose at this temperature also served to differentiate fecal from nonfecal coliforms. The fecal coliform strains tested were capable of producing gas as an end product of the fermentation of lactose at 44.5°C (Fig. 2), and they could take up atmospheric oxygen during the metabolism of lactose under aerobic conditions (Fig. 1). In contrast, nonfecal coliforms could not evolve gas from lactose under fermentative conditions (Fig. 2), and, furthermore, lactose was not metabolized aerobically, as evidenced by the lack of oxygen uptake at the elevated temperature (Fig. 1). In addition, nonfecal coliforms actively growing in lactose at 35°C were found to discontinue cell
346
DOCKINS AND McFETERS
APPL. ENVIRON. MICROBIOL. NOT SHIFTED (35C)
/ (-
SHIFTED I
-.IL--- ---
SHIFT
07
35
C-44.5-C/
0 0.6
ED~~~~, 0.5
0.4
-
0.
/
30
60
90 MINUTES
120
150
180
FIG. 4. Effect of a 35 to 44.5°C temperature shift upon growth of a nonfecal coliform in TSY broth (without glucose) containing 1% lactose.
division within 1 h after a shift to 44.5°C (Fig. 4). E. coli possesses two inducible, soluble, membrane-associated formate dehydrogenase complexes (24). One of these complexes is involved in the evolution of carbon dioxide and molecular hydrogen from formate, and it is functional only under anaerobic growth conditions (9, 22). If formate dehydrogenase is a target of the inhibitory effect of the elevated temperature upon nonfecal coliforms, the lack of gas production in EC broth at 44.5°C would be expected, and this was observed. However, aerobic metabolism which is conducted via metabolic pathways not involving formate dehydrogenase was also inhibited at 44.50C. Therefore, whether or not formate dehydrogenase is affected by the elevated temperature, additional metabolic sites of temperature sensitivity must also exist. There is much information in the literature concerning the effects of temperature fluctuations upon the composition and function of the E. coli cell membrane. Marr and Ingraham (19)
and others (4) have shown that E. coli increases the ratio of unsaturated to saturated fatty acids as growth temperature is increased and that the rate of change in this ratio is greatest at temperatures over 40°C. These closely regulated changes in the composition and the resultant physical properties of the lipid phase of the membrane have been found to affect many membrane-associated functions, including the lactose, f8-glucoside, and amino acid transport systems (3). Data collected in this study implicate cell membrane involvement in the observed temperature effects with respect to the expression of the fecal coliform phenotype. The use of radioactively labeled substrate demonstrated that a temperature increase from 35 to 44.50C drastically reduced the rates of ['4C]glucose uptake in nonfecal coliforms while those of fecal coliforms were essentially unchanged. We suggest that the inability of the nonfecal coliforms to incorporate ['4C]glucose or utilize lactose at 44.5°C could be due in part to deleterious effects
VOL. 36, 1978
FECAL COLIFORM ELEVATED-TEMPERATURE TEST
347
of the elevated temperature upon membrane of EC medium alone exerted a marked inhibitory lipid synthesis or upon control mechanisms in- effect upon the respiration rate of fecal coliforms volved in lipid phase transitions in this group of at 44.5°C; however, when combined with the other components of the highly buffered EC coliform organisms. Several factors led to the investigation of a broth this effect was essentially compensated for second elevated-temperature effect involving an and was not likely an important consideration aspect of lactose metabolism in nonfecal coli- (unpublished data). After entry into the cell via forms. The nonfecal coliforms tested seemed a specific permease-mediated transport system capable of metabolizing TSY broth at a minimal (14), the /-1,4 linkage of lactose is hydrolyzed level at 44.5°C, with evidence for this appearing via the enzyme fi-galactosidase. Because this in both the manometric experiments (Fig. 3) and enzyme is not used and in fact its synthesis is in the [14C]glucose incorporation experiments repressed during the metabolism of glucose (18), (Table 1). In contrast, nonfecal coliforms were it seemed possible that the additional inhibitory found to gradually cease cell division after a 35 effect of the elevated temperature upon the to 44.5°C shift in incubation temperature when nonfecal coliform may be focused upon this engrowing in TSY broth (without glucose) contain- zyme. Our results with cell-free extracts showed ing lactose (Fig. 4) and were incapable of metab- that the total 8-galactosidase activity was conohlzing the lactose-containing EC broth at that sistently greater in fecal coliforms than in nonfetemperature (Fig. 1). The bile salts component cal coliforms. By comparison, levels of aldolase activity, a constitutive enzyme chosen for its central position in carbon metabolism, were sim.30 r ilar for fecal and nonfecal coliforms (Table 2). In addition, thermal inactivation of fi-galactosidase in both fecal and nonfecal coliforms appeared at .25 a relatively low temperature compared with most mesophilic enzymes. Generally, the reacJE' tion rates of these enzymes approximately dou2 .20 ble for each 100C (Qlo = 2) rise in temperature c until the temperature of optimal activity is reached. Beyond this temperature (usually 55 to 600C) thermal inactivation results in a relatively .1 5 rapid loss of activity (16). Between 35 and 44.5°C the ,B-galactosidase-specific enzyme activity decreased rapidly (Fig. 5) as evidenced by a Qlo value of less than 1 (Table 2). In contrast, the activity of fecal and nonfecal coliform aldolase increased between 35 and 44.50C in all cases (Qio 55 > 1). The additive effect of the relatively low thennal inactivation temperature and the low , ---.--levels of ,8-galactosidase activity at temperatures over 35 to 380C resulted in virtually no meas-----------------30--- 4024. 10 20 30 40 50 urable fi-galactosidase activity at 44.50C in the TEMPERATURE nonfecal coliforms tested. A dramatic decrease FIG. 5. Effect of temperature upon 13-galactosidase activity in sonically disrupted cell-free extracts of of fl-galactosidase activity due to thermal inacfecal and nonfecal coliforms. Symbols: 0, fecal coli- tivation at temperatures over 35 to 380C was form, IMViC ++--; U, fecal coliforn, IMViC also observed for fecal coliforms (Fig. 5), but -+--; 0, nonfecal coliforn, IMViC -+++; 0, nonfe- there was sufficient enzyme activity present at cal coliforn, IMViC -+-+. 44.50C to allow the active metabolism of lactose. c
'
0
49
.10
-
.I0!
0
2:
I.
0
CC)
TABLE 2. Comparison of temperature effects on aidolase and 13-galactosidase activities offecal and
nonfecal coliforms
Coliformn
IMViC ++-E. coli B
IMViC -+++ IMViC-+-+
Fecal coliform phenotype + + -
-
Aldolse-specific enzyme activity 350C 44.5°C Q1oa 5.74 7.07 5.44 7.40
8.21 8.26 8.36 7.82
a Q,o, Activity at 44.50C per activity at 350C (10°C/9.5°C).
1.50 1.23 1.62 1.11
enzyme activity fi-Galactosidase-specific 35°C 44.50C
QIO"
0.175 0.041 0.006 0.021
0.043 0.016 0.005 0.001
0.26 0.41 0.87 0.05
348
DOCKINS AND McFETERS
This decrease in activity in fecal coliforms has been indirectly observed by Warren et al. (25) who found that lowering the 44.5°C incubation temperature by 1 or 20 resulted in significantly faster rates of ONPG hydrolysis. Two cellular sites of sensitivity to the elevated temperature in nonfecal coliforms have been identified in this study. Although incubation at 44.5°C may produce other effects deleterious to metabolism and growth of nonfecal coliforms, the inhibitory action upon cell membrane function and f,-galactosidase activity may be most important because of their initial position in lactose metabolism and the presence of that growth substrate in the commonly used differential media. Because of the heterogeneous nature of the coliform group of bacteria, it is quite possible that our findings will not explain all phenotypes observed in response to the elevated temperature; however, our results seem to be indicative of the response of the majority of these organisms Methods for separating fecal from nonfecal coliforms could be developed based upon the knowledge of these two temperature effects. One rapid spectrophotometric technique has been proposed by Warren et al. (25) and is based upon enzymatic hydrolysis of 0.06 M ONPG by fecal coliforms at 44.50C. Although much work remains to be done before they could be adopted for general use, such techniques could prove to be not only more rapid but more reliable than those relying upon gas production from lactose at 44.5°C. ACKNOWLEDGMENTS We thank Anne K. Camper and Susan B. Olson for technical aaaistance and John E. Schillinger and David G. Stuart for their helpful discuions and review of this manuscript. This research was supported by funds from the U.S. Department of the Interior under the Water Resources Research Act of 1964, Public Law 88-379 and administered through the Montana Joint Water Resources Research Center (grant OWRR A-099 Mont.).
1.
2.
3. 4.
5.
LITERATURE CITED American Public Health Association. 1976. Standard methods for the examination of water and wastewater, 14th ed. American Public Health Assoc., Washington, D.C. Bagley, S. T., and R. J. Seidler. 1977. Significance of fecal colifonn-positive Klebsiella. Appl. Environ. Microbiol. 33:1141-1148. Cronan, J. E., Jr., and E. P. Gelman 1975. Physical properties of membrane lipids: biological relevance and regulation. Bacteriol. Rev. 39:232-256. Cronan, J. E., Jr., and P. R. Vagelos. 1972. Metabolism and function of the membrane phospholipids of Ewcherichia coli. Biochim. Biophys. Acta 265:25-60. Eijkman, C. 1904. Die Garungprobe bei 460 als Hilfmittel bei der Trinkwasseruntersuchung. Zentralbl.
APPL. ENVIRON. MICROBIOL. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 37:742-752. 6. Geldreich, E. E., H. F. Clark, P. W. Kabler, C. B. Huff, and R. H. Bordner. 1958. The coliform group. I. Reactions in EC medium at 45 C. Appl. Microbiol. 6:347-349. 7. Geldreich, E. E., C. B. Huff, R. H. Bordner, P. W. Kabler, and H. F. Clark. 1962. The faecal coli-aerogenes flora of soils from various geographical areas. J. Appl. Bacteriol. 25:87-93. 8. Geldreich, E. E., B. A. Kenner, and P. W. Kabler. 1964. Occurrence of coliforms, fecal coliforms, and streptococci on vegetation and insects. Appl. Microbiol. 12:63-69. 9. Gray, C. T., and H. Gest. 1965. Biological formation of molecular hydrogen. Science 148:186-192. 10. Hajna, A. A., and C. A. Perry. 1938. A modified Eijkman medium for the isolation of Escherichia coli from sewage. Sewage Works J. 10:261-263. 11. Hajna, A. A., and C. A. Perry. 1943. Comparative study of presumptive and confirmatory media for bacteria of the coliform group and for fecal streptococci. Am. J. Public Health 33:550-556. 12. Hendricks, C. W. 1970. Formic hydrogenlyase induction as a basis for the Eijkman fecal coliform concept. Appl. Microbiol. 19:441-445. 13. Jagannathan, V., K. Singh, and M. Damodaran. 1956. Carbohydrate metabolism in citric acid fermentation. IV. Purification and properties of aldolase from Aspergillus niger. Biochem. J. 63:94-105. 14. Kennedy, E. P. 1970. The lactose permease system of Escherichia coli, p. 49-92. In J. Beckwith and D. Zipser (ed.), The lactose operon. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 15. Lederberg, J. 1950. The beta-D-galactosidase of Escherichia coli, strain K-12. J. Bacteriol. 60:381-391. 16. Lehninger, A. L 1975. Biochemistry, 2nd ed. Worth, New York. 17. Lowry, 0. H., N. J. Rosebrough, A. L Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 18. Magasanik, B. 1970. Glucose effects: inducer exclusion and repression, p. 189-219. In J. Beckwith and D. Zipser (ed.), The lactose operon. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 19. Marr, A. G., and J. L Ingraham. 1962. Effect of temperature on the composition of fatty acids in Escherichia coli. J. Bacteriol. 84:1260-1267. 20. Mishra, R. P., S. R. Joshi, and P. V. R. C. Panicker. 1968. An evaluation of the stindard biochemical and elevated temperature tests for differentiatig faecal and non-faecal coliforms. Water Res. 2:575-585. 21. Perry, C. A., and A. A. Hajna. 1944. Further evaluation of EC medium for the isolation of coliform bacteria and Escherichia coli. Am. J. Public Health Nat. Health 34:735-748. 22. Quist, R. G., and J. L. Stokes. 1969. Temperature range for formic hydrogenlyase induction and activity in psychrophilic and mesophilic bacteria. Antonie van Leeuwenhoek J. Microbiol. Serol. 35:1-8. 23. Rickenberg, H. V., C. Yanofsky, and D. M. Bonner. 1953. Enzymatic deadaption. J. Bacteriol. 66:683-687. 24. Ruiz-Herrera, J., A. Alvarez, and I. Figueroa. 1972. Solubilization and properties of fornate dehydrogenases from the membrane of Escherichia coli. Biochi. Biophys. Acta 289:254-261. 25. Warren, L S., R. E Benoit, and J. A. Jessee. 1978. Rapid enumeration of fecal coliforms in water by a colorimetric ,B-galactosidase assay. Appl. Environ. Microbiol. 35:136-141.
Vol. 36, No. 2
Printed in U.S.A.
Fecal Coliform Elevated-Temperature Test: a Physiological Basis WILLIAM S. DOCKINS AND GORDON A. McFETERS* Department of Microbiology, Montana State University, Bozeman, Montana 59717 Received for publication 14 March 1978
The physiological basis of the Eijkman elevated-temperature test for differentiating fecal from nonfecal coliforms was investigated. Manometric studies indicated that the inhibitory effect upon growth and metabolism in a nonfecal coliform at 44.50C involved cellular components common to both aerobic and fermentative metabolism of lactose. Radioactive substrate incorporation experiments implicated cell membrane function as a principal focus for temperature sensitivity at 44.5°C. A temperature increase from 35 to 44.50C drastically reduced the rates of [14C]glucose uptake in nonfecal coliforms, whereas those of fecal coliforms were essentially unchanged. In addition, relatively low levels of nonfecal coliform ,B-galactosidase activity coupled with thermal inactivation ofthis enzyme at a comparatively low temperature may also inhibit growth and metabolism of nonfecal coliforms at the elevated temperature. Some waterborne coliforms not normally considered to be of fecal origin are able to ferment lactose at 44.50C (6, 12, 20), and under certain conditions these organisms may be present in relatively high numbers in water samples (2). These findings constitute a basis for controversy over the validity of the elevated-temperature test for indicating the potential presence of pathogens in the aquatic environment. The widespread use and sanitary implications of the elevated-temperature procedure in detecting fecal water pollution stress the importance of determining and understanding the physiological mechanisms involved in colifonn growth and metabolism at 44.50C. A knowledge of the biochemical basis would also be of value in the formulation of improved fecal coliforn assay procedures. The primary objectives of this investigation were to locate the cellular site(s) of temperature sensitivity of nonfecal coliforms which might account for their failure to ferment lactose or glucose at 44.50C and to describe the physiological characteristics of coliforms insofar as they relate to the elevated-temperature test. MATERIALS AND METHODS
The coliform group of bacteria has been defined as "all the aerobic and facultative anaerobic, gram-negative, nonsporeforming, rodshaped, bacteria that ferment lactose with gas formation within 48 hours at 35C" (1). This group has long been used as an indicator of fecal contamination of natural waters. Because waterborne coliforms can originate from sources other than the intestinal tract of warm-blooded animals, such as from the soil or the surface of vegetation and insects (7, 8), it is desirable to determine the source of these organisms before attempting to relate them to fecal contamination and the potential presence of pathogens. A number of different procedures have been devised to separate fecal from nonfecal coliforms; however, the most successful and widely accepted technique involves incubation of coliforms at elevated temperatures. The first elevated-temperature test proposed by Eijkman (5) differentiated fecal from nonfecal coliforms by the ability of fecal coliforms to ferment glucose in a glucose-peptone broth at 460C. The current and most widely used elevated-temperature procedure relies upon fecal coliform fermentation of lactose in a highly buffered medium, with the production of hydrogen and carbon dioxide at 44.50C. These improved procedures are largely the result of the early work done by Perry and Hajna (10, 11, 21) and more recently by Geldreich et al. (6, 7). These workers and Mishra et al. (20) have reported a high correlation of gas production from lactose at 44.50C with coliforms that are normally associated with fecal pollution.
Cultures. The enteric bacterial cultures used in these studies were obtained from the Montana State University (MSU) Culture Collection or the American Type Culture Collection or were isolated by membrane filtration from streams in the area of Bozeman, Mont. All organisms conformed to the coliform designation as defined by the American Public Health Association (1). Stream isolates were further differentiated by indole, methyl red, Voges-Proskauer, and citrate (IMViC) classification and by the ability of the orga341
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DOCKINS AND McFETERS
nism to ferment lactose at 44.5°C in EC broth (Difco Laboratories) with the production of gas. Those coliforms capable of fermenting lactose with gas production at 44.5°C were termed fecal coliforms (1) and included Escherichia coli B (MSU culture collection no. 164) and stream isolates of IMViC types ++-(five strains) and -+--. Coliforms unable to ferment lactose at the elevated temperature (nonfecal coliforms) included Klebsiella pneumoniae ATCC 13883 and stream isolates of IMViC types -+++, -+-+, and -+--. All cultures were maintained on nutrient agar (Difco) and stored at refrigeration temperatures. Manometric methods. Cells employed in experiments with EC medium were grown in tryptic soy broth (Difco)-0.3% yeast extract-0.25% glucose (TSY) at room temperature and were aerated by shaking. The coliforms were harvested after 18 h by centrifugation at 3,000 x g (Sorvall, RC2-B), washed twice, resuspended in cold standard phosphate buffer (1), and standardized to 0.60 absorbance at 660 nm (A60) with a Varian Tectron model 635 spectrophotometer. Cells used in experiments in which TSY broth was the medium used were grown without shaking at 35°C in TSY broth. These cells were harvested after 18 h, washed once, and resuspended in cold phosphate buffer to Ass of 0.75. Single side-arm flasks (Gilson) were used in all manometric experiments. In experiments measuring 02 uptake, 0.5 ml of 40% KOH and a fluted filter paper wick were inserted into the center wells of each flask. Aliquots (4 ml) of the specified medium were added to the main chamber, and 1 ml of a cell suspension was placed in the side arm of each flask. Control flasks underwent similar preparation except that 1 ml of phosphate buffer was added to the side arm in place of the cell suspension. The flasks were attached to a Gilson differential respirometer and allowed to equilibrate at 44.50C (±0.20C, measured with standard thermometer) for approximately 20 min. In experiments that were done anaerobically, the respirometer flasks were purged of 02 by shaking them for 15 min before the equilibration time as nitrogen gas was passed through them. The 1-ml cell suspension in the side arm was then tipped into the main chamber, and the manometer readings were taken at 10- or 15-mm intervals. [14Cjglucose uptake. Coliforms used in ['4C]glucose uptake experiments were grown without shaking at 350C in TSY broth. The cells were harvested after 12 h, washed once with phosphate buffer, and resuspended in TSY broth to an Ass of 0.1 to 0.2. Flasks containing 60 ml of the cell suspension in TSY broth were equilibrated at 35 or 44.50C in water baths. At zero time 0.5 ml of uniformly labeled ['4C]glucose (0.5 ,uCi/ml, New England Nuclear Corp.) was added to the suspension, and a sample was taken immediately and at timed intervals thereafter. Aliquots (3 ml) of the culture suspension were removed for biomass determination (Ass), and 10-ml aliquots were filtered through a 0.45-,um filter (Millipore Corp.) for cellular radioactive glucose uptake measurements. Filters were washed with distilled water to remove extracellular ['4C]glucose, dried for 15 min at 105°C, and placed in poly Q scintillation vials (Beckman Instrument Co.). Toluene (4 ml) and 9 ml of Aquasol (New England
APPL. ENVIRON. MICROBIOL. Nuclear) were added to each vial. Labeled carbon counts and external standards were measured for each vial with a liquid scintillation counter (Beckman LSC 100) set at 5% error. Background counts were determined by filtering 10 ml of TSY broth and preparing this sample in the same manner as described above. Because of the similarity of the external standard counts, data were directly compared and expressed as counts per minute per absorbance unit (cpm/A6w). The maximal rate of uptake during the first 40 min of incubation was graphically calculated by determining the slope of the steepest sustained portion of the curve (counts per minute per A6s versis minutes). Temperature shift. Coliform organisms were grown at 35°C with shaking in TSY broth (without glucose) containing sodium acetate at a concentration of 1.0%. Cells were harvested after 18 h, washed twice with cold phosphate buffer, and resuspended in fresh TSY broth (without glucose). The A6s of this suspension was adjusted to fall within the range of 0.1 to 0.2. The suspension was equally divided into two flasks which were placed into a 35°C water bath and allowed to equilibrate for approximately 20 min. Aliquots (10 ml) of 10% lactose were added to each flask. The Ass of each cell suspension was measured at 15-min intervals for 3 h. After 1 h one flask was removed from the 35°C bath and placed at 44.5°C while the other flask remained at 35°C. ,B-Galactosidase assay. Coliform organisms were grown in TSY broth (without glucose) to which lactose at a final concentration of 1.0% was added. These cultures, fully induced for ,B-galactosidase, were harvested after 18 h, washed once, and resuspended in cold phosphate buffer. The A6s of the cell suspensions was adjusted to 1.25, and the cells were broken with a Bronwill Biosonic IV sonic oscillator set at 90% of maximum intensity. Sonication time was 15 min and the temperature of the suspension was not allowed to exceed 20°C. This method of sonication resulted in approximately 98% cell death. The extracts were then centrifuged at 3,000 x g for 5 min to remove remaining cells and larger cell fragments. The total protein contained in each extract was determined by the method of Lowry et al. (17). All cell-free extracts were stored on ice until use. f-Galactosidase activity was measured in the sonic extracts by a modified o-nitrophenyl-,8-D-galactopyranoside (ONPG) hydrolysis method (15,23). A 2.0-ml aliquot of 1.0 mM ONPG was added to 5 ml of the extract, and the mixture was incubated at a prescribed temperature for exactly 10 or 20 min. At the end of the incubation period, 2.0 ml of 0.5 M NaCO3 was added, and A420 was measured. In all assays the ONPG used was equilibrated to the temperature of the assay before addition. Results are expressed as A420 per minute per milligram of protein, and each data point is the average of three values. Aldolase assays. The preparation procedure for sonic extracts used in the aldolase assays was the same as that used for the f,-galactosidase assays except that tris(hydroxymethyl)aminomethane buffer (0.05 M, Sigma Chemical Co.) was substituted for phosphate buffer. Aldolase assays were performed by a modification of the spectrophotometric method of Jagannathan et al. (13). Aliquots (2 ml) of hydrazine sulfate
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FECAL COLIFORM ELEVATED-TEMPERATURE TEST
(3.5 mM in 0.1 mM ethylenediaminetetraacetic acid) were placed into a cuvette and allowed to equilibrate to 35 or 44.5°C in a spectrophotometer equipped with a recorder and a jacketed cuvette holder attached to a circulating heater. Fructose 1,6-diphosphate (0.10 ml, 12 mM) was then added, and the change in Ass over a 3-min interval was measured and recorded. Data were expressed as the initial slope of the Ass versus time per milligram of protein in the sonic extract (Ams per minute per milligram of protein). Aldolase assays were performed in triplicate, and each data point presented is the average of three initial
slopes.
RESULTS The results of manometric experiments in EC broth utilizing both a fecal coliforrn isolate (IMViC ++--) and a nonfecal coliform isolate (IMViC-+++) are shown in Fig. 1 and 2. The fecal coliform was capable of using lactose under both fermentative and aerobic conditions at 44.50C. The nonfecal coliform did not evolve gas from lactose at 44.50C in the respirometer under anaerobic conditions and furthermore did not metabolize lactose aerobically, as evidenced by the lack of oxygen uptake. Respiration studies in TSY broth gave results similar to those in EC broth (Fig. 3). Coliforms capable of a positive reaction in EC broth at the elevated temperature (44.50C), including an IMViC type ++-- isolate and E. coli B, showed a high respiration rate in TSY broth at 44.50C, whereas two nonfecal isolates (IMViC-+-+ 200
and -+++) demonstrated a miniimal rate of respiration at that temperature. The nonfecal organisms, however, showed minimal respiration in TSY broth at 44.50C, in contrast to the lack Of 02 uptake that was observed in EC broth at that temperature. Labeled glucose uptake studies were done to examine the effect of the elevated temperature upon bacterial transport functions (Table 1). Both nonfecal and fecal coliform isolates were capable of incorporating [14C]glucose at 350C in TSY broth and the maximal rates of uptake (counts per minute per A6ws) at this temperature were similar for the organisms tested. At 44.50C the fecal coliform isolates transported [14C]glucose at a rate comparable to that observed at 350C. In sharp contrast to this, nonfecal cohlforms showed little uptake of the labeled glucose at 44.50C compared with the maximal uptake rate at 350C. The rates of glucose incorporation (counts per minute per A6w, per minute) shown in Table 1 are not directly comparable between each organism tested because different activities of labeled glucose were used in different experiments; however, the ratio of gluocose uptake at the two temperatures can be directly compared for the various coliforms examined. A shift in growth temperature from 35 to 44.50C was followed where a nonfecal coliform (IMViC, -+++) was growing in TSY broth containing lactose. This experiment demonstrated
v-* FECAL COLSFORW (IMViC++--) o-O NON-FECAL COLIFORM (IMVIC -+++)
50 w
a.
o
343
100
MINUTES
FIG. 1. Comparison of respiration rates for fecal and nonfecal coliforms in EC broth at 44.5°C.
344
DOCKINS AND McFETERS
APPL. ENVIRON. MICROBIOL.
250 FECAL COLIFORM (IMViC+ +-- ISOLATE) O--O NON-FECAL COLIFORM (IMVIC-+++ ISOLATE)
2001
150w 0 w
100
50
30
d'0
--9 0------Q--
MINUTES
0
o
180 0 -----o----° 6
240
210 eo- --a
-
-O
FIG. 2. Comparison of gas evolution rates (CO2 + H2) for fecal and nonfecal coliforms in EC broth at 44.50C. Respirometer flasks were gassed with 1 00% nitrogen for 15 min to attain fermentation conditions.
that the high temperature stopped active cell division (Fig. 4). The temperature of the shifted suspension equilibrated to 44.50C from 350C within 10 min. The first observable change in the growth rate occurred within 30 to 60 min after the shift, and active growth of the culture gradually ceased between 30 and 90 min. A second differential effect of the elevated temperature was observed which involved the enzyme f8-galactosidase of fecal and nonfecal coliforms. f8-Galactosidase assays were done at temperature increments of approximately 5°C throughout the range of 10 to 45°C. The levels of f1-galactosidase activity in sonic extracts of fully induced fecal coliform cultures were consistently higher than those seen in comparable preparations from nonfecal coliforms (Fig. 5). Optimal activity of this enzyme in freshly prepared sonic extracts of fecal coliforms typically occurred at 30 ± 2°C, and the activity decreased rapidly as the temperature increased above 35 to 380C. At 44.50C fecal coliform f3-galactosidase activity was 25 to 50% of the optimal activity; however, nonfecal coliform /3-galactosidase activity was generally low or not measurable at that temperature. f8-Galactosidase activity was measured over the 10 to 45°C temperature range
for two additional sonically disrupted cultures not shown in Fig. 5. An E. coli B culture was similar to the two fecal coliforms shown but was slightly lower than those organisms in the level of enzyme activity that was observed. A nonfecal K. pneumoniae culture showed little fl-galactosidase activity at any of the assay temperatures. Spectrophotometric assays for aldolase activity were performed on sonic extracts of fecal and nonfecal coliforms to study the effect of the elevated temperature on this enzyme and to determine whether the reaction kinetics were affected by increased temperatures in the same manner as fB-galactosidase. Results of the aldolase assays for fecal coliforms (IMViC ++-and E. coli B) and nonfecal coliforms (IMViC -+++ and -+-+ isolates) indicate little difference between the two types of coliforms (Table 2), and the activity of that enzyme increased with temperature between 35 and 44.5°C regardless of the coliform type. DISCUSSION Although the elevated-temperature test has been used successfully for many years and several studies have addressed the sanitary significance of the procedure, the physiological basis
FECAL COLIFORM ELEVATED-TEMPERATURE TEST
VOL. 36, 1978
345
35O *-*
FECAL COLIFORM (L.gj U) *-4 FECAL COLIFORM(IMVIC + +--) °-O NON-FECAL COLIFORM (IMVIC -+++) a-O NON-FECAL COLIFORM (IMVIC -+-+)
,b200 w
oID150 I
SO FJ l
I
0~~~~~~~~~~~~
l
~ 100 50
100~~~~~
30
90
so
1 20
1 50
MINUTES
FIG. 3. Comparison of respiration rates for fecal and nonfecal coliforms in TSY broth at 44.50C.
TABLE 1. Incorporation of '4C-labeled glucose by fecal and nonfecal coliforms in TSY broth at 35 and
44.50C cpm/A,, per % Uptake colOrganism IMViC Fecal iform
FC1 BR6 BR4 BR1 BR1O NF1 NF2960 T8
++-++-++-++-++--+++ -+-+
+ + + + + -
min at:
35°C 44.50C 52.0 63.0 38.3 32.5 46.7 42.9 60.0 46.6 76.7 110.0 92.5 18.5 85.0 8.9 65.7 8.8
at 44.50C/
% uptake at 35°C 1.21 0.85 0.92 0.78 1.43 0.20 0.10 0.13
underlying the test has received little attention. Hendricks (12) examined several Enterobacter species using manometric techniques and suggested that the effects of the elevated temperature are focused upon the formate dehydrogenase complex. He postulated that there are at least two distinct biochemical types of Enterobacter in the aquatic environment and that the results of the elevated-temperature test de-
pended upon whether or not a coliform possessed formate dehyrogenase activity at 44.50C. The manometric data and growth curves obtained in this study indicate that the fecal coliform phenotype is a manifestation of more than a temperature effect upon the activity of the formate dehydrogenase complex in coliform bacteria. Although the elevated-temperature test is defined in terms of fermentative gas production from lactose at 44.50C, results of this study indicated that aerobic metabolism of lactose at this temperature also served to differentiate fecal from nonfecal coliforms. The fecal coliform strains tested were capable of producing gas as an end product of the fermentation of lactose at 44.5°C (Fig. 2), and they could take up atmospheric oxygen during the metabolism of lactose under aerobic conditions (Fig. 1). In contrast, nonfecal coliforms could not evolve gas from lactose under fermentative conditions (Fig. 2), and, furthermore, lactose was not metabolized aerobically, as evidenced by the lack of oxygen uptake at the elevated temperature (Fig. 1). In addition, nonfecal coliforms actively growing in lactose at 35°C were found to discontinue cell
346
DOCKINS AND McFETERS
APPL. ENVIRON. MICROBIOL. NOT SHIFTED (35C)
/ (-
SHIFTED I
-.IL--- ---
SHIFT
07
35
C-44.5-C/
0 0.6
ED~~~~, 0.5
0.4
-
0.
/
30
60
90 MINUTES
120
150
180
FIG. 4. Effect of a 35 to 44.5°C temperature shift upon growth of a nonfecal coliform in TSY broth (without glucose) containing 1% lactose.
division within 1 h after a shift to 44.5°C (Fig. 4). E. coli possesses two inducible, soluble, membrane-associated formate dehydrogenase complexes (24). One of these complexes is involved in the evolution of carbon dioxide and molecular hydrogen from formate, and it is functional only under anaerobic growth conditions (9, 22). If formate dehydrogenase is a target of the inhibitory effect of the elevated temperature upon nonfecal coliforms, the lack of gas production in EC broth at 44.5°C would be expected, and this was observed. However, aerobic metabolism which is conducted via metabolic pathways not involving formate dehydrogenase was also inhibited at 44.50C. Therefore, whether or not formate dehydrogenase is affected by the elevated temperature, additional metabolic sites of temperature sensitivity must also exist. There is much information in the literature concerning the effects of temperature fluctuations upon the composition and function of the E. coli cell membrane. Marr and Ingraham (19)
and others (4) have shown that E. coli increases the ratio of unsaturated to saturated fatty acids as growth temperature is increased and that the rate of change in this ratio is greatest at temperatures over 40°C. These closely regulated changes in the composition and the resultant physical properties of the lipid phase of the membrane have been found to affect many membrane-associated functions, including the lactose, f8-glucoside, and amino acid transport systems (3). Data collected in this study implicate cell membrane involvement in the observed temperature effects with respect to the expression of the fecal coliform phenotype. The use of radioactively labeled substrate demonstrated that a temperature increase from 35 to 44.50C drastically reduced the rates of ['4C]glucose uptake in nonfecal coliforms while those of fecal coliforms were essentially unchanged. We suggest that the inability of the nonfecal coliforms to incorporate ['4C]glucose or utilize lactose at 44.5°C could be due in part to deleterious effects
VOL. 36, 1978
FECAL COLIFORM ELEVATED-TEMPERATURE TEST
347
of the elevated temperature upon membrane of EC medium alone exerted a marked inhibitory lipid synthesis or upon control mechanisms in- effect upon the respiration rate of fecal coliforms volved in lipid phase transitions in this group of at 44.5°C; however, when combined with the other components of the highly buffered EC coliform organisms. Several factors led to the investigation of a broth this effect was essentially compensated for second elevated-temperature effect involving an and was not likely an important consideration aspect of lactose metabolism in nonfecal coli- (unpublished data). After entry into the cell via forms. The nonfecal coliforms tested seemed a specific permease-mediated transport system capable of metabolizing TSY broth at a minimal (14), the /-1,4 linkage of lactose is hydrolyzed level at 44.5°C, with evidence for this appearing via the enzyme fi-galactosidase. Because this in both the manometric experiments (Fig. 3) and enzyme is not used and in fact its synthesis is in the [14C]glucose incorporation experiments repressed during the metabolism of glucose (18), (Table 1). In contrast, nonfecal coliforms were it seemed possible that the additional inhibitory found to gradually cease cell division after a 35 effect of the elevated temperature upon the to 44.5°C shift in incubation temperature when nonfecal coliform may be focused upon this engrowing in TSY broth (without glucose) contain- zyme. Our results with cell-free extracts showed ing lactose (Fig. 4) and were incapable of metab- that the total 8-galactosidase activity was conohlzing the lactose-containing EC broth at that sistently greater in fecal coliforms than in nonfetemperature (Fig. 1). The bile salts component cal coliforms. By comparison, levels of aldolase activity, a constitutive enzyme chosen for its central position in carbon metabolism, were sim.30 r ilar for fecal and nonfecal coliforms (Table 2). In addition, thermal inactivation of fi-galactosidase in both fecal and nonfecal coliforms appeared at .25 a relatively low temperature compared with most mesophilic enzymes. Generally, the reacJE' tion rates of these enzymes approximately dou2 .20 ble for each 100C (Qlo = 2) rise in temperature c until the temperature of optimal activity is reached. Beyond this temperature (usually 55 to 600C) thermal inactivation results in a relatively .1 5 rapid loss of activity (16). Between 35 and 44.5°C the ,B-galactosidase-specific enzyme activity decreased rapidly (Fig. 5) as evidenced by a Qlo value of less than 1 (Table 2). In contrast, the activity of fecal and nonfecal coliform aldolase increased between 35 and 44.50C in all cases (Qio 55 > 1). The additive effect of the relatively low thennal inactivation temperature and the low , ---.--levels of ,8-galactosidase activity at temperatures over 35 to 380C resulted in virtually no meas-----------------30--- 4024. 10 20 30 40 50 urable fi-galactosidase activity at 44.50C in the TEMPERATURE nonfecal coliforms tested. A dramatic decrease FIG. 5. Effect of temperature upon 13-galactosidase activity in sonically disrupted cell-free extracts of of fl-galactosidase activity due to thermal inacfecal and nonfecal coliforms. Symbols: 0, fecal coli- tivation at temperatures over 35 to 380C was form, IMViC ++--; U, fecal coliforn, IMViC also observed for fecal coliforms (Fig. 5), but -+--; 0, nonfecal coliforn, IMViC -+++; 0, nonfe- there was sufficient enzyme activity present at cal coliforn, IMViC -+-+. 44.50C to allow the active metabolism of lactose. c
'
0
49
.10
-
.I0!
0
2:
I.
0
CC)
TABLE 2. Comparison of temperature effects on aidolase and 13-galactosidase activities offecal and
nonfecal coliforms
Coliformn
IMViC ++-E. coli B
IMViC -+++ IMViC-+-+
Fecal coliform phenotype + + -
-
Aldolse-specific enzyme activity 350C 44.5°C Q1oa 5.74 7.07 5.44 7.40
8.21 8.26 8.36 7.82
a Q,o, Activity at 44.50C per activity at 350C (10°C/9.5°C).
1.50 1.23 1.62 1.11
enzyme activity fi-Galactosidase-specific 35°C 44.50C
QIO"
0.175 0.041 0.006 0.021
0.043 0.016 0.005 0.001
0.26 0.41 0.87 0.05
348
DOCKINS AND McFETERS
This decrease in activity in fecal coliforms has been indirectly observed by Warren et al. (25) who found that lowering the 44.5°C incubation temperature by 1 or 20 resulted in significantly faster rates of ONPG hydrolysis. Two cellular sites of sensitivity to the elevated temperature in nonfecal coliforms have been identified in this study. Although incubation at 44.5°C may produce other effects deleterious to metabolism and growth of nonfecal coliforms, the inhibitory action upon cell membrane function and f,-galactosidase activity may be most important because of their initial position in lactose metabolism and the presence of that growth substrate in the commonly used differential media. Because of the heterogeneous nature of the coliform group of bacteria, it is quite possible that our findings will not explain all phenotypes observed in response to the elevated temperature; however, our results seem to be indicative of the response of the majority of these organisms Methods for separating fecal from nonfecal coliforms could be developed based upon the knowledge of these two temperature effects. One rapid spectrophotometric technique has been proposed by Warren et al. (25) and is based upon enzymatic hydrolysis of 0.06 M ONPG by fecal coliforms at 44.50C. Although much work remains to be done before they could be adopted for general use, such techniques could prove to be not only more rapid but more reliable than those relying upon gas production from lactose at 44.5°C. ACKNOWLEDGMENTS We thank Anne K. Camper and Susan B. Olson for technical aaaistance and John E. Schillinger and David G. Stuart for their helpful discuions and review of this manuscript. This research was supported by funds from the U.S. Department of the Interior under the Water Resources Research Act of 1964, Public Law 88-379 and administered through the Montana Joint Water Resources Research Center (grant OWRR A-099 Mont.).
1.
2.
3. 4.
5.
LITERATURE CITED American Public Health Association. 1976. Standard methods for the examination of water and wastewater, 14th ed. American Public Health Assoc., Washington, D.C. Bagley, S. T., and R. J. Seidler. 1977. Significance of fecal colifonn-positive Klebsiella. Appl. Environ. Microbiol. 33:1141-1148. Cronan, J. E., Jr., and E. P. Gelman 1975. Physical properties of membrane lipids: biological relevance and regulation. Bacteriol. Rev. 39:232-256. Cronan, J. E., Jr., and P. R. Vagelos. 1972. Metabolism and function of the membrane phospholipids of Ewcherichia coli. Biochim. Biophys. Acta 265:25-60. Eijkman, C. 1904. Die Garungprobe bei 460 als Hilfmittel bei der Trinkwasseruntersuchung. Zentralbl.
APPL. ENVIRON. MICROBIOL. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 37:742-752. 6. Geldreich, E. E., H. F. Clark, P. W. Kabler, C. B. Huff, and R. H. Bordner. 1958. The coliform group. I. Reactions in EC medium at 45 C. Appl. Microbiol. 6:347-349. 7. Geldreich, E. E., C. B. Huff, R. H. Bordner, P. W. Kabler, and H. F. Clark. 1962. The faecal coli-aerogenes flora of soils from various geographical areas. J. Appl. Bacteriol. 25:87-93. 8. Geldreich, E. E., B. A. Kenner, and P. W. Kabler. 1964. Occurrence of coliforms, fecal coliforms, and streptococci on vegetation and insects. Appl. Microbiol. 12:63-69. 9. Gray, C. T., and H. Gest. 1965. Biological formation of molecular hydrogen. Science 148:186-192. 10. Hajna, A. A., and C. A. Perry. 1938. A modified Eijkman medium for the isolation of Escherichia coli from sewage. Sewage Works J. 10:261-263. 11. Hajna, A. A., and C. A. Perry. 1943. Comparative study of presumptive and confirmatory media for bacteria of the coliform group and for fecal streptococci. Am. J. Public Health 33:550-556. 12. Hendricks, C. W. 1970. Formic hydrogenlyase induction as a basis for the Eijkman fecal coliform concept. Appl. Microbiol. 19:441-445. 13. Jagannathan, V., K. Singh, and M. Damodaran. 1956. Carbohydrate metabolism in citric acid fermentation. IV. Purification and properties of aldolase from Aspergillus niger. Biochem. J. 63:94-105. 14. Kennedy, E. P. 1970. The lactose permease system of Escherichia coli, p. 49-92. In J. Beckwith and D. Zipser (ed.), The lactose operon. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 15. Lederberg, J. 1950. The beta-D-galactosidase of Escherichia coli, strain K-12. J. Bacteriol. 60:381-391. 16. Lehninger, A. L 1975. Biochemistry, 2nd ed. Worth, New York. 17. Lowry, 0. H., N. J. Rosebrough, A. L Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 18. Magasanik, B. 1970. Glucose effects: inducer exclusion and repression, p. 189-219. In J. Beckwith and D. Zipser (ed.), The lactose operon. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 19. Marr, A. G., and J. L Ingraham. 1962. Effect of temperature on the composition of fatty acids in Escherichia coli. J. Bacteriol. 84:1260-1267. 20. Mishra, R. P., S. R. Joshi, and P. V. R. C. Panicker. 1968. An evaluation of the stindard biochemical and elevated temperature tests for differentiatig faecal and non-faecal coliforms. Water Res. 2:575-585. 21. Perry, C. A., and A. A. Hajna. 1944. Further evaluation of EC medium for the isolation of coliform bacteria and Escherichia coli. Am. J. Public Health Nat. Health 34:735-748. 22. Quist, R. G., and J. L. Stokes. 1969. Temperature range for formic hydrogenlyase induction and activity in psychrophilic and mesophilic bacteria. Antonie van Leeuwenhoek J. Microbiol. Serol. 35:1-8. 23. Rickenberg, H. V., C. Yanofsky, and D. M. Bonner. 1953. Enzymatic deadaption. J. Bacteriol. 66:683-687. 24. Ruiz-Herrera, J., A. Alvarez, and I. Figueroa. 1972. Solubilization and properties of fornate dehydrogenases from the membrane of Escherichia coli. Biochi. Biophys. Acta 289:254-261. 25. Warren, L S., R. E Benoit, and J. A. Jessee. 1978. Rapid enumeration of fecal coliforms in water by a colorimetric ,B-galactosidase assay. Appl. Environ. Microbiol. 35:136-141.
