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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1980, p. 739-742 Vol. 30, No.

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0099-2240/8004-0739/04$02.000

Staphylococcus aureus in Rural Drinking Watert


MARK W. LECHEVALLIER AND RAMON J. SEIDLER*
Department ofMicrobiology, Oregon State University, Corvallis, Oregon 97331

Coagulase-positive Staphylococcus aureus was isolated from over 6% of 320


rural drinking water specimens. Well water was the most common source exam-
ined. The presence of S. aureus was not found to correlate with the presence of
coliform bacteria. Strains of Staphylococcus that produced enterotoxin A were
found in 40% of the samples containing S. aureus. Additional studies showed that
faucet aerator screens were common sources of high cell densities of S. aureus.
Staphylococcus aureus is one of the most by two procedures. In the first, sterile sample bottles
common agents of food poisoning. In addition, it were distributed to residents in a rural area who ob-
is the agent of a variety of skin abscesses, pus- tained their water from individual wells. Residents
tules and, less frequently, septicemia, enterocol- were instructed to take samples after running the
itis, osteomylitis, and pneumonitis (13). This water for several minutes. The bottles were collected
3 to 4 h after being filled and stored on ice until
pathogen is widely distributed, having been iso- cultured within the next 1 to 3 h. The second proce-
lated from human nasal passages and skin (22), dure involved sampling water specimens sent to the
human and animal feces (18), dust and moisture Oregon State University Department of Microbiology
droplets (23), clothing (11), food, and the diges- water laboratory. These samples were usually mailed
tive tract of flies (16). One study traced the in and had a maximum 30-h transit time limit before
origin of a food poisoning incident to contami- sampling.
nated water used to cool hard-boiled eggs (7). In In a separate series of studies, the investigators
that outbreak S. aureus contaminated the eggs, went into homes and collected specimens from the
multiplied, and produced enterotoxin from an same faucet by three procedures. First, the faucet
screens were removed with sterile forceps. The screens
initial level of 100 organisms per ml of water. were washed in 100 ml of buffered dilution water (1)
S. aureus in dfinking water may also serve as contained in a sterile, wide-mouth collection bottle. In
a source for colonizing residents exposed to con- the second procedure, the faucet end near the location
taminated water. Shinefield and his colleagues of the removed screen was sterilized by brief heat with
(19), in their investigation of bacterial interfer- a propane torch. The water in the household pipes
ence, found that they could set up a carrier state was then sampled after flowing for about 5 s. In the
in the nose of 50% of newborn infants by inocu- last procedure, fresh water from the storage tank was
lating 200 to 400 cocci. Other investigators found sampled after flowing at maximum capacity through
that the inoculum required to induce infection the faucet for some 4 min with the screen removed.
in traumatized skin was very small. Colonization Media. Approximately 70 ml of sample was filtered
onto a 0.45-,tm membrane filter (Gelman Instrument
occurred in the majority ofsites when inoculated Co., Ann Arbor, Mich.). The membrane filter was
with a few hundred cells, and some infections placed onto Staphylococcus 110 medium (Difco Lab-
occurred after an inoculation of approximately oratories, Detroit, Mich.) and incubated at 350C for
10 cells (15). 48 h. Typical staphylococcal colonies were inoculated
During routine monitoring of bacteria found into coagulase plasma (Difco), and the coagulase re-
in rural drinking water, S. aureus was occasion- action was interpreted according to the method of
ally isolated on standard plate count agar. Be- Sperber and Tatini (20). Coagulase-positive bacteria
cause of its unexpected presence, S. aureus was were further identified as S. aureus by oxidase test,
catalase test, Gram stain, and morphology. The ability
specifically monitored in subsequent samples of the bacteria to ferment glucose and mannitol an-
and compared with numbers of standard plate aerobically was determined by using the media and
count bacteria and the presence of total coli- procedures recommended by the Subcommittee on
forms. Isolates of S. aureus were also tested for Taxonomy of Staphylococci and Micrococci (21).
enterotoxin production, and an attempt was Thermostable nuclease activity was determined by
made to determine their origin in drinking water the method of Lachica et al. (14). Hydrolysis of deoxy-
supplies. ribonucleic acid was assayed on deoxyribonuclease
(DNase) test agar (Difco). The presence or absence of
MATERIALS AND METHODS total coliforms was detected by the five-tube most-
Sample collection. Water samples were obtained probable-number technique and processing through
the completed step (1). Heterotrophic bacteria were
(Difco) (1).
tTechnical paper 5383, Oregon Agricultural Experiment enumerated on standard plate count agarenterotoxins
Station. Enterotoxin assay. Staphylococcal
739
740 LECHEVALLIER AND SEIDLER APPL. ENVIRON. MICROBIOL.
A, B, and C were obtained from M. S. Bergdoll, Food sample 29 prevented detection of S. aureus. S.
Research Institute, Madison, Wis. The antiserum was aureus occurred in the water in the pipes behind
obtained from the Foods and Nutrition Department, the screens in samples 48 and 49 and in the
Oregon State University. The enterotoxins were as- storage tankwell water in sample 14. S. aureus
sayed by the microslide technique described by Cas- was also isolated from a kitchen faucet aerator
man et al. (6).

RESULTS TABLE 1. Incidence of S. aureus in rural drinking


water supplies
Table 1 summarizes the results of the surveys. No. with No. with
Three samplings were made; the first two were No. of typical coagu- % of sam-
collected from a rural area in western Oregon, Sample pe-
riod samples staphylo- lase-posi- ples with
and the third was received by mail. The first tested coccal tive reac- S. aureus
sampling contained 48 water samples, of which colonies tion
14 had typical staphylococcal colonies when enu- 1 48 14 4 8.3
merated on Staphylococcus 110 medium. Of 2 49 12 2 4.1
these 14 samples, 4 contained S. aureus species 3 223 118 14 6.3
based on positive reactions in coagulase, DNase Total 320 144 20 6.25
test agar, fermentation of glucose and mannitol,
and cell morphology. The second sampling con- TABLE 2. Numbers of S. aureus and total bacteria
sisted of 49 specimens, of which 12 yielded typ- in relation to coliforms and enterotoxin production
ical staphylococcal colonies. Two contained S. Typical Con- Entero-
aureus. The third collection consisted of 223 Sam- staphy- Average firmed Coagu- toxin A
samples, of which 118 yielded typical staphylo- ple lococci total plate col- lase re-
coccal colonies. Of the 118 samples, 14 contained no. /100 count/ml forms action produc-
S. aureus. In the total of 320 samples collected, ml forms ~~~tion
S. aureus was recovered with a frequency of 17 1 1.0 x 102 - + +
48 4 2.0 x 102 - + -
6.25%. 49 12 <10 - + (83)a +
Coliforms were detected in 49 of the 320 sam- 67 38 10 - + -
ples. Only five times were coliforms and S. au- 14 2 50 - + -
reus simultaneously detected (Table 2). 29 >600 1.9 x 104 - + (25) -
1152 1 NS - + +
Of the 20 samples that had S. aureus in the 1162 3 5.2 x 102 - + +
water (Table 2), 8 had strains that produced 1169 3 >3.0 x 103 - + +
enterotoxin A. Only three enterotoxins were as- 1195 205 2.6 x 103 - + (77) -
sayed for (A, B, and C), so it is possible that 1199 159 3.1 x 102 + + (25) +
some of the isolates produced other toxins. All
1203 6 7.5 x 102 - + -
1209 13 >3.0 x 103 - + -
the S. aureus strains produced coagulase en- 1223 >330 3.4 x 103 + + (30) +
zymes, thermostable nuclease, and DNase. None 1296 4 30 - + -
of these tests therefore uniquely correlated with 1316 4 >3.0 x 103 + + +
enterotoxin production. 1318 1 >3.0 x 103 + + -
1353 1 <10 - + -
To determine the origin(s) of S. aureus con- 1559 27 >3.0 x 103 - + (48) -
tamination, samples were collected from three 1365 >375 >3.0 x 103 + + (50) -
points in household water supply systems (Table a Numbers in brackets are the estimated percentage of
3). All the aerator screens had high bacterial coagulase-positive S. aureus in the sample based on the testing
counts. The high counts in all specimens of of at least 10 colonies from each specimen.

TABLE 3. Origin of S. aureus in rural drinking water


Storage water House lines Aerator screen

stypical
staphy o- Coagulase reac-
tion
No. of typi-
cal staphylo- Coagulase re-
action
No. of typical
staphylococci
Coagulase
action
re-
cocci/100 ml cocci/100 ml
14 27 + (11)0 NSb Confluent NDc
29 >300 - NS Confluent ND
48 32 - 21 + (24) 129 + (50)
49 15 - 97 + (50) Confluent ND
9 0 NS 0 NS 15 + (47)
See Table 2.
bNS, No sample available.
c ND, Not determined.
VOL. 39, 1980 STAPHYLOCOCCUS IN DRINKING WATER 741
screen in a home that had no detectable staph- aureus was also occasionally isolated from water
ylococci in the water supply (sample 9). drawn from house lines and the storage tank.
The latter isolates could not have emanated
DISCUSSION from colonized focal points near or around the
Private dfinking water supplies are rarely if faucet screen since this was sterilized with a
ever routinely examined for microbiological torch before sampling. The implication is that S.
quality. As a result, consumption of contami- aureus may be contaminating the well or has
nated rural drinking water has a low chance of colonized the storage tank or house lines. In
being associated with a waterbome illness. In such cases, the faucet screen surfaces could have
addition, many bacteriological and viral agents provided the necessary nutritional conditions to
of gastroenteritis produce self-limiting illness promote regrowth or provided the focal point
with symptoms much like the flu. Such is the for Staphylococcus deposition as contaminated
case with intoxications caused by toxin-produc- water flowed through the screen.
ing strains of S. aureus which induced nausea, In addition to allowing colonization by S. au-
vomiting, and diarrhea. As a result of these facts, reus, faucet aerator screens are also known to be
little definitive evidence is available to estimate the focal point for the colonization of opportun-
the incidence of waterborne outbreaks in rural istic pathogens of other bacterial genera. For
homes served by private sources of water. example, Cross et al. demonstrated that Pseu-
In the present study, over 6% of the rural domonas species could be isolated from water
drinking water samples contained S. aureus. on faucet aerators (8), and others have isolated
There was no correlation between the simulta- Flavobacterium species (10, 12). Frequent clean-
neous presence of the coliform indicator group ing of faucet screens, flushing of storage tanks,
and S. aureus. Therefore, results from coliform and, in cases of severe contamination, intermit-
analyses alone would not have been an entirely tent chlorination of rural water supplies would
satisfactory measure of rural dfinking water help eliminate various contaminants from the
quality. The extended survival ability of staph- water and therefore lower the possibility of wa-
ylococci may explain their presence when coli- terborne illness.
forms cannot be detected (9). A better correla- It would certainly be relevant to determine
tion existed between densities of total plate the incidence of staphylococci in other rural
counts and S. aureus. Sixty-three percent of the water supplies where groundwater quality and
samples containing S. aureus had plate counts climatic conditions differ from those in the pres-
exceeding 300/ml whereas only 19% not contain- ent study area.
ing S. aureus had similar counts.
There are many reasons for potential concern ACKNOWLEDGMENTS
when S. aureus is present in domestic drinking We appreciate the assistance of M. J. Woodburn and T. N.
water supplies. One report has already described Morita, Department of Food and Nutrition, Oregon State
for their help in identifying staphylococcal enter-
food poisoning resulting from contaminated wa- University,
otoxins. Pam Lytz performed the coliform analyses on some
ter used to cool hard-boiled eggs (7). To induce of the samples.
food intoxication, only a small amount of enter- This study was supported by a grant from the Water
otoxin need be produced (2, 3, 5, 17). Bergdoll Resources Research Institute at Oregon State University.
(4) has indicated that 1 ug or less of enterotoxin LITERATURE CITED
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