ENVR 133 - Environmental Health Microbiology - Spring

Detection of Fecal Streptococci and Enterococci by Multiple Tube and Membrane Filter Methods

The fecal streptococci and enterococci are members of the genera Streptococcus and
Enterococcus. These bacteria are spherical, gram positive and grow in chains. Most are
facultative, "aerotolerant" anaerobes and have no aerobic metabolism. They must ferment
sugars and are unable to synthesize heme, a necessary component of cytochromes. The
enterococci and many fecal streptococci are normal flora of the gastrointestinal tract, where they
are present at high concentrations. However, they can sometimes spread from the
gastrointestinal tract and cause frank disease. Bacteremia, wound infections, urinary tract
infections and endocarditis are known to occur. These illnesses are more likely in newborns and
in the elderly. Enterococci are now a concern in nosocomial infections as well as infections of
non-hospitalized populations because of growing antibiotic resistance, especially to antibiotics
such as vancomycin.

The streptococci and enterococci are relatively fastidious in their growth requirements because
they have lost the ability to synthesize many essential nutrients. Therefore, they require several
amino acids and the B vitamins to be grown in the laboratory. The fecal streptococci/enterococci
will grow: (1) in high NaCl (6.5%), i.e., salt tolerant; (2) in 1% methylene blue, (3) in bile-esculin
medium (hydrolyze esculin in the presence of bile); and (4) at pH 9.6.

The streptococci/enterococci can be classified on the basis of:

(i) Lancefield (cell wall) antigens (A, B, C, D, E-T)
(ii) colony morphology and hemolysis,
(iii) biochemical reactions
(iv) resistance to physical and chemical agents,
(v) antigenic composition and serological reactions;
(vi) ecology
(vii) Molecular genetic properties

The fecal streptococci and enterococci belong to serogroup D. Serogrouping of the

streptococci is based on the group carbohydrate of the cell wall. Most group D streptococci are
nonhemolytic or alpha hemolytic on sheep RBCs. (beta hemolysis = complete hemolysis; alpha
hemolysis = partial hemolysis). Not all group D streptococci are enterococci; they differ from
enterococci by inability to grow in 6.5% NaCl and inhibition by 40% bile. There are more than 2
dozen species of enterococci, some of which colonize or infect humans and other animals and
some of which occur naturally in the environment (some do both). The important enterococci
with respect to human fecal contamination are E. faecalis, E. faecium and E. durans.
Non-enterococci, esp. S. bovis and S. equinus are also in the gut. The enterococci utilize PYR
(L-pyrrolidonyl-beta-napthylamide) as a substrate (possess the enzyme pyrrolidonly
arylamidase); streptococci will not; the test is rapid (few minutes).

Detection and enumeration of enterococci was difficult because some standard media for fecal
streptococci (KF agar and m-enterococcus agar and azide dextrose broth) detected organisms
of non-fecal origin and organisms less likely to be of human origin (e.g., S. bovis and S.
equinus). Standard media for the fecal streptococci include: KF agar, m-enterococcus agar,
GTC agar, citrate azide agar, azide dextrose broth and PSE (esculin-azide medium).
Azide-containing media have several disadvantages: deficiencies in detecting injured cells,
selectivity against S. bovis, S. equinus and S. avium and a longer incubation period (48 hours).
GTC medium has given high false-positive rates from environmental samples. Like the
limitations of coliform tests, some of the bacteria detected in the fecal streptococci tests are not

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restricted to the feces of warm-blooded animals. For example, E. faecalis subsp. liquefaciens is
found on vegetation and in insects and certain soils.

The term enterococci is used by some as synonymous with the term fecal streptococci. Indeed,
the taxonomy of the "fecal streptococci" is now based on the name "enterococci". In the U.S.A.
the term enterococci as used by the U.S. Environmental Protection Agency is meant to include
only E. faecalis and E. faecium and to exclude S. bovis, S. equinus and the Group Q
streptococci. A medium was developed that was intended to specifically detect the enterococci
by a membrane filter method (Levine et al., 1975). The advantage of this test is that it excludes
some streptococci that are more likely to be associated with non-human fecal contamination,
and hence is considered a more human-specific fecal indicator. However, this contention is
somewhat flawed because some of the excluded streptococci, such as S. bovis, have been
found in human feces. Furthermore, the test for enterococci does not exclude some subspecies
of E. faecalis (e.g., liquefaciens) that are not always associated with fecal contamination, and it
detects species of enterococci are also found naturally in the environment, such as E. mundtii
and E. casseliflavus. Hence, it is not strictly a feces-specific medium.

Epidemiological and microbiological studies in the U.S.A. demonstrated that concentrations of

enterococci in swimming waters correlated with risks of gastrointestinal illness in swimmers
(Cabelli et al., 1982; 1983). The results of these studies led the U.S. EPA to recommend
enterococci as the bacterial criterion for the acceptability of swimming waters (U.S. EPA, 1985).
More recent studies in the UK and elsewhere led to the World Health Organization
recommending the use of “intestinal enterococci” as the preferred indicator of fecal
contamination of recreational waters. Intestinal enterococci are defined as a subgroup of the
fecal streptococci that can grow at 44oC, hydrolyze MUG in the presence of thallium acetate,
nalidixic acid and 2,3,5-triphenlttetrazolium chloride in specified liquid media.

The development of new membrane filter media, the best of which is now called mEI medium,
greatly improved the ability to detect primarily the enterococci. In the U.S these organisms are
defined as the true "enterococci. However, mEI does not detect only E. faecalis and E. faecium,
because as previously noted, some fecal streptococci will grow on mEI medium. However, this
may not be a major deficiency because other species of enterococci have been detected in
human as well as animal feces..

Another major improvement in the detection of enterococci has been the development of a
defined substrate medium, "Enterolert"® (Idexx Corp.). The medium contains a defined
substrate consisting of 4-methyl-umbelliferyl Beta-D-glucoside. When this substrate is
hydrolyzed by the Beta-D-glucosidase produced by the enterococci, it produces 4-methyl
umbelliferone as a blue fluorescent hydrolysis product, as well as the D-glucose product that
can be used as a substrate for bacterial growth. Furthermore, the method provides results in 24
hours. The improved Enterococcus membrane filter test also provides results in 24 hours. The
multiple tube tests for enterococci require 48-72 hours to provide confirmed results (Abbott et
al., 1998; Budnick et al., 1996; Eckner, 1998).

PURPOSE

To detect fecal streptococci and enterococci in selected samples of water and wastewater using
multiple tube and membrane filter methods.

Two multiple tube methods will be tested, one for fecal streptococci and one for enterococci:

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Fecal streptococci: Conventional two-step method using azide dextrose broth followed by
conformation on bile-esculin-azide agar
Enterococci: Enterolert one-step method for using defined substrate medium

Two membrane filter methods will be tested: one for fecal streptococci and one for enterococci
Fecal streptococci: conventional method using m-Enterococcus agar, followed by confirmatory
testing for growth on bile esculin agar and the hydrolysis of pyrrolidonyl-beta-naphthylamide
(PYR). The PYR test detects the enterococci among the fecal streptococci.
Enterococci: one-step, 24-hour test using mEI medium.

MATERIALS

Petri plates of m-enterococcus agar for detection of fecal streptococci.

Petri plates of modified mEI agar for detection of enterococci.
Tubes of azide dextrose broth
Plates of bile esculin azide agar
PYR reagent kit
Enterolert dehydrated medium, in ampoules for 100-ml volumes.
Standard Methods buffered water as diluent for Enterolert medium, 300-ml volumes per group
Sterile 16x150 mm culture tubes, with slip caps, 25 per group
Milk dilution bottles of peptone water, 99 ml per bottle.
Squirt bottles of peptone water; to rinse membrane filter funnels.
Standard membrane filter apparatus consisting of filter support base, filter
funnel, vacuum manifold and vacuum source.
Membrane filters, 0.45 um pore size, 47 mm diameter, gridded.
Pipets
Incubators and water baths
Sterile, flat-blade forceps
Water and wastewater samples. To be specified by the instructor.

PROCEDURE

Sample Dilution:
Obtain water or wastewater sample designated by the instructor.
Dilute the sample serially 10-fold as specified by the instructor.
Shake samples 25 times immediately prior to dilution.
Make 10-fold dilutions by adding 11 ml of sample to 99 ml of diluent in a milk dilution bottle.
Shake 25 times.

Membrane Filtration:
Obtain plates of m-enterococcus agar
Starting with the greatest sample dilution, filter two 20-ml volumes of sample (diluted or
undiluted) through separate membrane filters.
Place one membrane filter on each of the test media: KF streptococcal agar and mEI agar
Repeat the membrane filtration and plating procedure with the next most dilute sample as just
described, until all sample dilutions have been membrane-filtered and plated.
Incubate m-enterococcus plates inverted at 35oC for 48 hours.
Incubate mEI plates inverted at 41oC for 24 hours

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After the incubation periods, observe and count the colonies of fecal streptococci (m-
enterococcus agar) and enterococci (mEI agar) and record the results for the sample dilutions
analyzed.

NOTE: Fecal streptococci colonies are dark red to pink. Enterococci colonies are dark blue or
purple with a dark precipitate on the medium below the colony (hint: turn the plate over to see
the medium under the colony).

Multiple Tube Test - Conventional Method

Place 25 tubes of azide dextrose broth with slip caps in a 5 x 5 array in a test tube rack and
them for the 5 sample volumes (dilutions) to be inoculated.
Starting with the greatest sample dilution, inoculate 1-ml volumes into each of 5 tubes.
Repeat with the next most dilute sample until all sample dilutions have been inoculated.
Incubate the tubes at 35oC for 24 hours
Observe for growth (turbidity). If a tube shows growth (turbidity) it is a presumptive positive. If a
tube shows no growth/turbidity at 24 hours, incubate another 24 hours and re-examine for
growth (48 hours total incubation).
Inoculate a loopful of culture from positive tubes onto bile esculin azide agar by streaking onto a
plate (according to the instructor's directions).
Incubate plates of bile esculin azide agar at 35oC for 24 hours
After incubation, observe bile esculin azide agar plates for characteristic growth of fecal
streptococci: brownish-black colonies with brown halos. These are confirmed fecal streptococci
To determine which fecal streptococci are enterococci, test the bacterial growth on bile esculin
azide agar using the PYR test, following the directions provided by the instructor.

Enterolert: Multiple Well Test in Quantitrays

Dispense 100 ml of sample dilution into a sterile bottle as directed by the instructor
Aseptically, add a100-ml ampoule of dehydrated Enterolert medium to the 100 ml of sample
water and swirl or shake to dissolve.
Pour the contents of each bottle into a Quantitray and seal the tray.
Incubate the trays at 41oC for 24 hours.
After the incubation period, expose the trays to long wavelength UV light and observe for blue
fluorescence. Blue fluorescence is a positive result; no fluorescence is a negative result.
Record the number of positive and negative wells of each size in the Quantitray.

RESULTS AND QUESTIONS

Using the data for the whole class, determine the concentrations of fecal
streptococci and enterococci in each water sample by each method of analysis.

For the membrane filter methods, use the membrane filter colony counts of the most countable
dilution(s). Remember that the desired counts are in the range of 20-80 or 20-60 colonies per
filter. In some cases you will have to use lower colony counts because higher numbers of
colonies were not detected in any of the sample dilutions
plated. Express concentrations as colonies of each organism per 100 ml.
Remember to correct for the volume filtered (20 ml) and the sample dilution.

For the multiple tube method, compute the MPN value and the upper and lower 95% confidence
limits per 100 ml of sample using the MPN table and the "best" three sample dilutions of the 5
dilutions tested. Typically, this is the last (greatest) dilution with 5/5 positive tubes and the next

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two dilutions after that. If no dilution gave 5/5 positives, use the dilution with the most positive
tubes and the next two dilutions after that.

For the Enterolert Quantitrays, use the numbers of positive wells out of the total of each size to
find the MPN value and 95% confidence interval in the table provided by the manufacturer.

Bring your data to class for compiling it with the data for the rest of the class.

Compute the mean colony counts and 95% confidence limits per 100 ml for each indicator in
each water or wastewater sample, using the computation procedures previously used for total
and fecal coliforms and E. coli.

What are the relative concentrations of each indicator organism in each sample and are the
relationships consistent for different samples?

Which indicator is highest in concentration and which is lowest in concentration?

Because the enterococci are a subgroup of the fecal streptococci, do the latter outnumber the
former? By what extent? Is this consistent for the different samples?

What is the extent of reduction of each indicator by the sewage treatment plant. You can
determine this by calculating the relative amount of each indicator in the effluent when the
amount in the raw sewage is considered 100%. This will give you the percent of initial indicator
bacteria remaining in the effluent. Which indicator is most persistent? Which is least
persistent?

For each indicator, what is its "background" level in Morgan Creek (the upstream level) and
what is the impact of the sewage treatment plant effluent on the Creek? This analysis requires
you to compare the levels of each indicator in the upstream water, the effluent and the
downstream water.

REFERENCES

Abbott, S., B. Caughley, and G. Scott. 1998. Evaluation of Enterolert for the Enumeration of
Enterococci in the Marine Environment. New Zealand Jour. of Marine & Freshwater
Research. 32: 505-513.
APHA. 9230. Fecal Streptococcus and Enterococcus Groups. In: Standard Methods for the
Examination of Water and Wastewater, 20th edition. APHA, AWWA, WEF, Washington, DC.
Budnick, G.E., R.T. Howard and D.R. Mayo. 1996. Evaluation of Enterolert for Enumeration of
Enterococci, In Recreational Waters. Appl. Environ. Microbiol. 62: 3881-3884.
Cabelli V J. Dufour A P. Mccabe L J. Levin M A. Swimming Associated Gastroenteritis and Water
Quality. American Journal of Epidemiology 115 (4). 1982. 606-616.
Cabelli V J., Dufour A P.,McCabe L. J., Levin M A. A MARINE RECREATIONAL WATER
QUALITY CRITERION CONSISTENT WITH INDICATOR CONCEPTS AND RISK
ANALYSIS. Journal - Water Pollution Control Federation 55 (10). 1983. 1306-1314
Eckner, K.F. 1998. Comparison of Membrane Filtration and Multiple-Tube Fermentation by the
Colilert and Enterolert Methods for Detection of Waterborne Coliform Bacteria, Escherichia
coli and Enterococci Used in Drinking and Bathing Water Quality Monitoring in Southern
Sweden. Appl. Environ. Microbiol. 64: 3079-3083.
Levin M A. Fischer J R. Cabelli V J. Membrane Filter Technique for Enumeration of Enterococci
in Marine Waters. Applied Microbiology 30 (1). 1975. 66-71.

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Murray, B.E. (1990) The life and times of the Enterococcus. Clin Microbiol Rev., 3(1): 46–65.
US EPA (1997) Method 1600: Membrane Filter Test Method for Enterococci in Water. EPA-
821-R97-004. Office of Water, Washington, DC (http://www.epa.gov/nerlcwww/1600.pdf)
Wade, T.J., RL. Calderon, E. Sams, M. Beach, K. P. Brenner, A.H. Williams and A.P. Dufour
(2006) Rapidly Measured Indicators of Recreational Water Quality Are Predictive of
Swimming-Associated Gastrointestinal Illness. Env. Health Perspectives, 114(1):24-28.
World Health Organization (2003) Guidelines for Safe Recreational Water Environments.
Volume 1: Coastal and Fresh Waters. World Health Organization, Geneva.
Downloadable at: http://www.who.int/water_sanitation_health/bathing/srwe1/en/